Enzyme substrates for visualizing acidic organelles

ABSTRACT

The present invention relates to the visualization of acidic organelles based upon organelle enzyme activity. The organelle substrates of the invention are specific for enzyme activity of the organelle and label these organelles, such as lysosomes, rendering them visible and easily observed. Substrates of the present invention include substrates that produce a fluorescent signal. The fluorogenic acidic organelle enzyme substrates of this invention are designed to provide high fluorescence at low pH values and are derivatized to permit membrane permeation through both outer and organelle membranes of intact cells and can be used for staining cells at very low concentrations. They can be used for monitoring enzyme activity in cells at very low concentrations and are not toxic to living cells or tissues.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of co-pending U.S. applicationSer. No. 12/381,560, filed Mar. 11, 2009.

This invention was made with Government support under grant5R43MH079542-02 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the visualization of acidic organellesbased upon organelle enzyme activity. The fluorescent organellesubstrates of the invention are specific for enzyme activity of theorganelle and label these organelles, such as lysosomes, rendering themfluorescent and easily observed.

BACKGROUND OF THE INVENTION

Acidic organelles are present in all cells and tissues of mammalian,plant, yeast and fungal cells, except red blood cells. Many bacteriaalso contain acidic compartments. These acidic organelles are ofteninvolved in metabolism and catabolism of foreign molecules that arebrought into the cell by endocytosis. They are often the first line ofdefense against foreign bacterial or viral infection. The acidic pH ofendosomes is critical to the process by which lipid-enveloped virusesenter the cytoplasm after their cellular uptake by receptor-mediatedendocytosis. Phagocytosis is the process where extra cellular particlessuch as bacteria, are engulfed in the cell and then fused to lysosomesfor digestion. Acidic organelles have also been shown to be responsiblefor digestion of high molecular weight proteins, oligosaccharides,glycolipids or peptides by the cell. In addition, they are ofteninvolved in therapeutic drug metabolism. Among the cellular organellesthat have been found to mediate their enzyme activities by acidificationare lysosomes, acidic endosomes, phagosomes, clathrin-coated vesiclesand Golgi vesicles.

Lysosomes are an example of an acidic cytoplasmic organelle. Lysosomeshave been found to be involved in a variety of cellular processesincluding repair of the plasma membrane, defense against pathogens,cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cellsignaling. To date, more than 50 acidic hydrolytic enzymes have beenidentified that are involved in ordered lysosomal degradation ofproteins, lipids, carbohydrates and nucleic acids. Functionaldeficiencies in these lysosomal enzymes, however, are indicative of anumber of disease states.

Many inherited carbohydrate metabolic diseases, especially lysosomalstorage diseases, have been identified to date. These diseases includeHurler disease (MPS IH, i.e., mucopolysaccharidosis type IH), Scheiedisease (MPS IS), Hurler-Scheie disease (MPS I H/S), Hunter disease (MPSII), Sanfilippo disease (MPS III), Morquio disease (MPS IV),Maroteaux-Lamy disease (MPS VI), Sly disease (MPS VIII), mannosidosis,fucosidosis, sialidosis, asparylglycosaminuria, Gaucher disease(glucosylceramide lipidosis), Krabbe disease(galactoceramide-lipidosis), Fabry disease, Schindler disease, GM1gangliosidoses, GM2 gangliosidoses, Tay-Sachs disease, Sandhoff disease,and mucolipidoses. As a group, these diseases are the most prevalentgenetic abnormalities of humans. Gaucher disease, Sandhoff disease,Krabbe disease, and Tay-Sachs syndrome comprise the majority of patientsin this category and are categorized as sphingolipidoses in whichexcessive quantities of undegraded fatty components of cell membranesaccumulate because of inherited deficiencies of specific catabolicenzymes within lysosomes.

The therapeutic options for treating these diseases are relativelylimited; in fact, there are currently no available therapies for many ofthese disorders. To date, therapeutic efforts have mainly focused onstrategies for augmenting enzyme concentrations to compensate for theunderlying defect. For this reason, new, sensitive and specific assaysfor monitoring lysosomal enzyme activities in living cells that will beof significant value in monitoring the success of current therapies andfor discovery of new therapeutic strategies for diseases of lysosomalorigin are needed.

Traditional lysosomal stains include the non-specific phenazine andacridine derivatives, neutral red and acridine orange, that areaccumulated in the acidic vesicles upon being protonated. Fluorescentlylabeled latex beads and macromolecules, such as dextran, can also beaccumulated in lysosomes by endocytosis in a variety of experiments.

Prior stains, methods and assays for visualizing acidic organelles suchas lysosomes are not useful for monitoring lysosomal enzyme activitiesin living cells. For example, weakly basic amines have been shown toselectively accumulate in cellular compartments with low internal pH.When further linked to chromogenic or fluorogenic probes, they can beused to label these compartments. Among these is the frequently usedacidotropic probe,N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl)methylamine,dihydrochloride (hereafter referred to as DAMP). DAMP is not itselffluorescent and fixation and permeabilization of the cell, followed bythe use of anti-DNP antibodies conjugated to a fluorophore, an enzyme orferritin are required in order to visualize the staining pattern. Thefluorescent dyes neutral red and acridine orange are also commonly usedfor staining acidic organdies, but they lack specificity and are notwell retained in the organelles, particularly after fixing andpermeabilization.

The compounds dansyl cadaverine and monodansyl cadaverine, which containan aliphatic amino groups for targeting to the lysosome have beendescribed as a lysosomotropic reagents. However, dansyl cadaverine isonly described as having an effect on the function of human naturalkiller cells and human polymorphonuclear leucocytes. More recentresearch describes monodansyl cadaverine as a fluorescent label, howeverit is described as useful as a label for autophagic vacuoles, as itfails to label either endosomal compartments or lysosomes. In addition,the dansyl fluorophore is excited in the ultraviolet region (<350 nm),which is generally incompatible with living systems, has a low quantumyield and has a low extinction coefficient (less than 5,000) requiringhigh concentrations of dye when staining cells.

In addition, certain dipyrrometheneboron difluoride fluorophores linkedto a weak base that is only partially protonated at neutral pH asdescribed in U.S. Pat. No. 5,869,689, have been used for generallabeling of lysosomes. But none of these probes are useful formonitoring specific enzyme activities in lysosomes or for metabolicanalyses or analysis of enzyme activity defects.

Of the numerous lysosomal storage assay systems that have been reported,the majority utilize either fluorescent (4-methylumbelliferyl)substrates, chromogenic (nitrophenolic glycosides), glycolipids labeledwith fluorescent dyes or radioactive substrates for detection oflysosomal glycosidase activities. These methods, however, utilize eithercell lysate from cells or tissue homogenates, HPLC separation ofenzymatic products and UV or fluorescent analysis or other presentinvention. Other enzymes which may be detected using the systems andsubstrates of the present invention will be obvious to a person skilledin the art.

5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluoresceindiphosphate, diammonium salt

A solution of5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluorescein (76 mg,0.147 mmoles) was dissolved in anhydrous pyridine (2.5 mL) underanhydrous N₂ and was cooled to 0° C. (ice-bath) and phosphorousoxychloride (100 uL, 1.07 mmole) added slowly with stirring. Thismixture was allowed to stir at 0° C. for 2 hours and at room-temperaturefor 18 hours. The reaction mixture was centrifuged, and the orange solidredissolved in ice-water (20 mL) and neutralized with dilute ammoniumhydroxide solution (pH 8). The resulting solution was lyophilized, andapplied to a column of Sephadex G-10 and eluted with water. Fractionscontaining the first quenching product to elute from the column werecombined, lyophilized to give a pale yellow solid, that was trituratedwith methanol and redried to give the title compound (33 mg, 34%). TLCanalysis (3:3:6:1 CH2CL2: MeOH: H2O: HOAc) (Rf=0.2) (quenching,non-fluorescent until acid treatment).

Example 7

complex analysis techniques. None of these assays, therefore, are welldesigned for an in vivo, or live-cell high-throughput systems detection,and require either biopsy or extensive cell preparation steps. Inaddition, the fluorescent dyes used in these assays are not amenable tothe low pH environment of the lysosome and therefore do not allowimaging in the lysosome in its native environment. Accordingly, nomethods have thus far been described that employ intact lysosomes or alive-cell format to monitor lysosomal enzyme activities.

SUMMARY OF THE INVENTION

The present invention includes methods and describes the synthesis ofmaterials for analysis of acidic organelle enzyme activities, whetherpresent in cells or in isolated cell-free organelle preparations, usingsubstrates that produce a visible signal when acted upon by suchenzymes. The method comprises: preparing a labeling solution containingan enzyme substrate or substrates of the present invention, where thelabeling solution comprises a marker that produces a visible signal atlow pH values and possessing a covalently attached basic amine moietyand is derivatized for specific enzyme analysis; and incubating with asample comprising isolated acidic organelles, or live cells or tissues,allowing the labeling solution a sufficient time to produce labeling ofthe acidic organelles. Substrates of the present invention includesubstrates that produce a fluorescent signal. The use of the enzymesubstrates of the invention are optionally combined with the use ofadditional detection reagents. The labeled cells are optionally observedusing a means for detecting a fluorescent signal for microscopicanalysis, fluorescence activated cell sorting or high-throughputscreening analyses.

The fluorogenic acidic organelle enzyme substrates of this invention aredesigned to provide high fluorescence at low pH values and arederivatized to permit membrane permeation through both outer andorganelle membranes of intact cells, can be used for staining cells atvery low concentrations and are not toxic to living cells or tissues.The instant substrates and methods are useful for investigatingmetabolism, investigating the biogenesis of lysosomes, investigating thedevelopment of autophagic vacuoles, fusion of phagosomes with acidiclysosomes, investigating retina and cultured neurons, monitoring changesin lysosomal enzyme activities, monitoring enzyme activities associatedwith diseases, evaluating the relative levels of enzymes in both normaland diseased states and detecting pH gradients within lysosomes. Inparticular, the instant substrates and methods are useful forinvestigating lysosomal glycosidase activity, lysosomal peptidaseactivity, lysosomal aryl sulfatase activity, lysosomal lipase activitylysosomal phosphatase activity, lysosomal esterase activity. The currentinvention is also useful for labeling non-mammalian cells that possessacidic organelles, including yeast, spermatozoa and plant cells.

Among the enzymes that are present in acidic organelles and that can bedetected using the substrates of the present invention areα-Mannosidase, β-Galactosidase, α-Galactosidase, β-Glucosidase,α-Glucosidase, b-Glucuronidase, β-acetylglucosaminidase, Neuraminidase,Hyaluronidase, Lipase, Phospholipase A, Esterase, Acid Phosphatase,Phospholipase C, Acid phospho-diesterase, Arylsulfatase A/B,Chondrosufatase, Lysozyme, β-Xylosidase, α- and β-Fuco-sidases,Cathepsin A, Acid Carboxy-Peptidase, Alanylaminopeptidase,Leucylaminopeptidase, Dipeptidase, Cathepsin B, Cathepsin H, CathepsinL, Cathepsin C, Dipeptidyl Aminopeptidase II, Cathepsin D, Cathepsin E,Collagenase, Renin, Kininogen activator, Plasminogen activator, andAspartylglucosyl aminidase. Those enzymes listed are given as adescriptive embodiment of the present invention but not intended to be acomplete list of possible enzyme activities which may be detected usingthe substrates and methods of the

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows fluorescein based staining probes of the present invention.

FIG. 2 shows Krabbe LysoMarker™ substrates of the present invention.

FIG. 3 shows coumarin based probes of the present invention.

FIG. 4 shows naphthofluorescein—based probes of the present invention.

FIG. 5 shows benoxazoylumbelliferyl substrates of the present invention.

FIG. 6 shows benzoxazolylcoumarin substrates of the present invention.

FIG. 7 shows gaucher substrates based on M1247 of the present invention.

FIG. 8 shows lysosomal targeted esterase substrates of the presentinvention.

FIG. 9 shows hexosaminidase (Tay-Sachs) lysosomal targeted substrates ofthe present invention.

FIG. 10 shows lipophilic targeting group esterase and glycosidasesubstrate syntheses of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention utilizes fluorogenic, chromogenic orchemiluminescent enzyme substrates for the labeling and tracing ofacidic organdies in cells and cell-free systems. These new substratesselectively accumulate in cellular compartments with low internal pH andcan be used to investigate the enzyme levels responsible forbiosynthesis, degradation and recycling of cellular components and formeasuring specific enzyme defects involved in a number of human diseaseslinked to enzyme activity in lysosomes within live cells.

The acidic organelle substrates of the present invention have thegeneral formula

T-F(R)-BLOCK

Where T represents a Targeting group that is a weakly basic aminecontaining compound that partitions the substrate to the acidicorganelle or lysosome, F represents a reporter that provides a visiblesignal upon the removal of BLOCK that has further elaboration withsubstituent or substituents R to provide for fluorescence at low pHvalues, and BLOCK represents a biological molecule including acarbohydrate, aminoacid, peptide, phosphate, sulfate, lipid or nucleicacid group that can be removed by specific enzyme activity within theacidic organelle or lysosome, thus allowing F to provide a visiblesignal.

In a particular embodiment, acidic organelle substrates of the presentinvention have the general formula:

T-LINK-F(R)-BLOCK(R′).

In this embodiment, the LINK portion of LINK-T is a covalent linkage,serving to attach a weakly basic amine, T, to the reporter, F. Anysuitable covalent linkage that does not interfere with the ability ofthe substrate to selectively accumulate in acidic organelles is anacceptable covalent linkage for the purposes of the present invention.In one embodiment, LINK is a single covalent bond. Preferred LINK groupshave 1-20 nonhydrogen atoms selected from the group consisting of C, N,O and S. Such LINK groups are composed of any combination of chemicalbonds, including ether, thioether, amine, ester, carboxamide,sulfonamide, hydrazide bonds, and single, double, triple carbon-carbonbonds, and aromatic or heteroaromatic bonds. Preferred LINK groups arecomposed of any combination of single carbon-carbon bonds andcarboxamide bonds. Selected specific examples of LINK optionally includemethylenes, oligomethylenes, phenylenes, thienyls, carboxamides, andsulfonamides. In one embodiment of the invention, LINK contains 1-6carbon atoms. In an additional embodiment of the invention, LINK has theformula —(CH₂)_(a)(CONH(CH₂)_(b))_(z)—, where a has any value from 0-5,b has any value from 1-5 and z is 0 or 1.

In this embodiment BLOCK group can be further modified with asubstituent or substituents (R′) that improve membrane permeability ofthe substrate through cellular membranes.

The substituent or substituents R, which may be the same or different,are selected from the group hydrogen, halogen, cyano, alkyl, substitutedmethane, perhalogenated alkyl, perfluoroalkyl, halomethyl, alkoxy,cycloalkyl, arylalkyl, acyl, aryl, heteroaryl, alkenyl or alkynyl; or aLINK-T moiety.

Preferably, the substituent or substituents R that are not a LINK-Tmoiety include electron-withdrawing groups such as, halogen, cyano,alkyl, aryl, heteroaryl, alkenyl or may be a hydrogen. More preferably,the substituent or substituents R that are not a LINK-T are halogen,aryl or cyano. Alternatively, for those substrates where R is linked toa fused aromatic 6-membered ring that is optionally and independentlysubstituted once or more, at any position, by halogen, cyano, alkyl,perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, alkylthio,alkylamido, amino, monoalkylamino, dialkylamino, carboxamide, hydroxy,mercapto, aryl, heteroaryl, aryl-amido, heteroaryl-amido, aryl-oxy,heteroaryl-oxy, aryl-amino, or heteroaryl-amino, or 1-2 additional fusedbenzo or heteroaromatic rings that are themselves optionally furthersubstituted by halogen, amino or carboxamide. Any of the fused aromatic6-membered rings or additional fused benzo or heteroaromatic rings isoptionally substituted one or more times by a LINK-T moiety.

As used herein, aryl is an aromatic or polyaromatic substituentcontaining 1 to 4 aromatic rings (each ring containing 6 conjugatedcarbon atoms and no heteroatoms) that are optionally fused to each otheror bonded to each other by carbon-carbon single bonds. Each aryl isbound by a single bond, and is optionally substituted as describedbelow.

As used herein, a heteroaryl is an aromatic group that contains at leastone heteroatom (a non-carbon atom within the ring structure). Eachheteroaryl is a single 5- or 6-member ring, or is a fused 2- or 3-ringstructure. The heteroaryl group contains one or more heteroatoms, e.g.as pyrrole, thiophene, or furan (single ring, single heteroatom), oroxazole, isoxazole, oxadiazole, or imidazole (single ring, multipleheteroatoms), or benzoxazole, benzothiazole, or benzimidazole(multi-ring, multiple heteroatoms), or benzofuran or indole (multi-ring,single heteroatom). Each heteroaryl is bound by a single bond, and isoptionally substituted as described below.

Any aryl, heteroaryl, aryl-amido, heteroaryl-amido, aryl-oxy,heteroaryl-oxy, aryl-amino or heteroaryl-amino substituent is optionallyand independently substituted one or more times by halogen, amino,carboxamide, hydroxy or mercapto.

Any of said alkenyl or alkynyl substituents independently has 2-6carbons, and is optionally substituted by halogen, alkyl, cyano,carboxylate ester, carboxamide, aryl, heteroaryl, or additional alkenylor alkynyl groups. Preferably an alkenyl group is an ethenyl, dienyl ortrienyl group.

Each of the alkyl substituents, as well as the alkyl portions of alkoxy,cycloalkyl, arylalkyl, alkylamino, alkylthio or alkylamido substituentsindependently has 1-6 carbons, and is optionally substituted by halogen,amino, alkylamino, dialkylamino, carboxamide, hydroxy, mercapto orcyano.

In a particular embodiment, at least one of R substituents is a LINK-Tmoiety, a LINK-T substituted methine, or one of the F substituents thatis a fused 6-membered ring is further substituted by a LINK-T moiety.For all embodiments, where F is substituted by more than one LINK-T,they are the same or different.

The targeting group T has the general formula —CR_(c)R_(d)—NR_(e)R_(f).The substituents R_(c) and R_(d) are independently hydrogen or alkylshaving 1-6 carbons that are linear or branched. Typically, R_(e) andR_(f) are hydrogen or alkyls having 1-16 carbons, more preferably R_(e)is methyl and R_(f) is either methyl, alkyl having 2-16 carbons, or arepart of a nitrogen heterocyclic ring system such as morpholine,piperidine, pyrrolidine, piperazine, imidazole, oxazepine, azepine orpyrrole. Where R_(e) or R_(f) are alkyl groups, each alkyl group isoptionally further substituted by halogen, carboxamide, oxy, hydroxy,mercapto or cyano. In addition, any alkyl group is optionally furthersubstituted by a primary, secondary or tertiary amine, where the alkylgroups present on the amine independently have 1-6 carbons. In anadditional embodiment of the invention, one of R_(e) and R_(f), whentaken in combination with the LINK moiety, forms a five- toeight-membered ring.

The amine substituents R_(e) and R_(f) are each independently H or alinear or branched alkyl having 1-6 carbons. Alternatively, R_(e) andR_(f), when taken in combination form a R_(e) and R_(f) that preservesthe basic nature of the amine nitrogen. Preferably, the nitrogenheterocycle is a pyrrolidine, a piperidine, a piperazine, morpholine, animidazole, an azepine (including diazepines and triazepines) or anoxazepine. In another embodiment of the invention, the aminesubstituents R_(e) and R_(f), when taken in combination withsubstituents R_(c) and R_(d), or with the LINK moiety, form a saturatedfive- or six-membered nitrogen heterocycle that is a substitutedpyrrolidine or piperidine.

For all embodiments of the invention, the basic targeting group moiety Tis optionally present in the form of a salt of a strong acid, forexample a hydrochloride salt, sulfate salt, perchlorate salt, or otherorganic acid salts.

Selected specific embodiments of substrates useful for the staining ofacidic organelles and lysosomes are described in FIGS. 1 through 10.

The substrates and probes of the present invention are readily preparedusing the methods described herein. Specific methods for preparing thecovalent linkage, LINK, and Targeting Group T are demonstrated in theExamples.

Compounds wherein the LINK or T moiety incorporates a cyclic structureare prepared by reaction of a preformed reactive reporter (F) with anappropriate amine-containing intermediate, or by preparingamine-containing pyrroles prior to formation of the reporter.

The substrates of the invention are only partially protonated at neutralpH. The spectral properties of the probe can be tuned over a wide rangeof the visible and near infra-red spectrum through selection of thesubstituents as described herein, making them especially useful formulticolor applications. Similarly, selection of substituents allow thepH selectivity of the substrate to be tuned for specific applications.For example, a substrate having a Targeting moiety T that is less basicwill be protonated by more acidic conditions, and therefore moreselectively accumulate at locations having a lower pH.

The substrates of the present invention are freely permeant to cellmembranes, and typically selectively accumulate in acidic organelles.The staining characteristics are generally not reversed or are onlypartially reversed by subsequent treatment of the cells with additionalweakly basic cell-permeant compounds. Accordingly, staining may bepreserved even after fixation and/or permeabilization of the cells.

The substrates of the present invention are utilized by preparing alabeling solution containing one or more of the substrates of thepresent application, introducing the labeling solution into the samplecontaining or thought to contain acidic organelles, incubating thesample for a time sufficient to produce a detectable visual signal, andobserving or analyzing the staining pattern in the sample. The samplemay be a cell or cells that contain acidic organelles or the sample maycontain isolated acidic organelles (i.e. not incorporated in a cell), orthe sample may be two solutions separated by a semi-permeable membrane.

The degree of staining of acidic organelles is a reflection of the pHgradient present across the acidic organelles membrane at the time ofstaining, i.e., the degree of staining is indicative of whether or notthe organelle is acidic at the time of staining. While the substrates ofthe present invention are typically used for staining the acidicorganelles of live cells, the present invention is also useful forstaining isolated (i.e. cell-free) acidic organelles, provided theorganelles are not disrupted and a pH gradient still exists between theorganelle and the medium in which it is suspended. While in general thepresence of acidic organelles can be considered an indicator of cellviability, it is possible to render a cell non-viable, while stillretaining acidic organelles in the sample.

Furthermore, the substrates of the present invention can be made fromfluorescent dyes that have the property of modifying their fluorescencespectrum as a function of pH upon enzyme turnover. Among the REPORTERdyes that exhibit changes in signal dependent upon the pH environment atthe site of activity are the coumarins, fluoresceins,naphthfluoresceins, carbocyanines and rhodamines.

The pure substrates of the present invention may have low solubility inwater. Typically a stock solution is prepared by dissolving a known massof the pure substrate in an organic solvent. Preferred organic solventsare DMSO, DMF, N-methylpyrrolidone, acetone, acetonitrile, dioxane,tetrahydrofuran, methanol or ethanol or other completely water-misciblesolvents. Alternatively, the substrate is dispersed in a waterimmiscible solvent or oil, or is evaporated from an organic solventleaving a thin film. The labeling solution is prepared by diluting analiquot of the stock solution into an aqueous or partially aqueousbuffer to the desired labeling concentration. In one embodiment of theinvention, two or more substrates of the invention are present in thelabeling solution, having similar or distinct spectral properties.

In general the amount of substrate added in the labeling solution is theminimum amount required to yield detectable staining of the acidicorganelles present in the sample within a reasonable time, with minimalbackground staining or staining of other organdies or cellularstructures. The amount of substrate required for staining eukaryoticcells depends on the sensitivity required for staining the intracellularacidic organelles, the number of cells present, the permeability of thecell membrane to the substrate, and the time required for the probe tolocalize to the organelles. The required concentration for the labelingsolution is determined by systematic variation in labeling concentrationuntil a satisfactory fluorescent labeling is accomplished. Typically,the amount of substrate required for staining animal cells is 10 to 200uM, preferably below 500 uM.

Low concentrations of substrate require longer incubation times forequivalent fluorescent brightness to be reached. Typically cellsincubated in 10 uM labeling solution require about 2 hours to reach anarbitrary level of staining that is reached in about 30 minutes using a200 uM labeling solution. For those embodiments where the acidicorganelles to be stained are vacuoles present in plant cells, yeast orother fungal cells, a higher concentration of substrate is used, due tothe lower permeability of the plant, yeast or other cell membranes.Typically, when staining fungal cells, a substrate concentration of 1 mMis satisfactory to give good vacuolar staining.

Staining concentrations less than about 100 uM give good staining ofacidic organelles in live animal cells. At higher concentrations ofstain, background fluorescence increase in live cells, but resolution ofacidic organelles after fixation is improved. Staining of isolated(cell-free) acidic organelles typically requires lower concentrations ofsubstrates.

The exact concentration of substrates to be used is dependent upon theexperimental conditions and the desired results and optimization ofexperimental conditions is required to determine the best concentrationof stain to be used in a given application. Such conditions andconcentrations needed for optimal staining can be readily discerned byone of skill in the art in view of the present disclosure.

The sample optionally comprises cell-free acidic organelles or cellsthat contain acidic organelles. Any cells that contain acidic organellescan be used, including but not limited to, fresh or cultured cells, celllines, cells in biological fluids, cells in tissue or biopsy, yeastcells, plant cells and sperm cells. Where the sample contains cells, thecells are optionally abnormal cells, such as tumor cells or other cancercells, where the abnormal cells are present in vitro or in vivo, orprimary cells derived from patients with specific disease. Acidicorganelles of interest that are stained using the present method ofstaining include, but are not limited to, lysosomes, phagovacuoles,endosomes, yeast vacuoles and acrosomes. In one embodiment of theinvention, the staining method is used to label all lysosomalcompartments in the sample. Typically, the acidic organelles that arestained are lysosomes or acrosomes. More typically, the acidicorganelles that are stained are lysosomes.

Most plant and fungal cells (including the unicellular fungi and yeast)contain one or more very large, fluid-filled vesicles called vacuoles.In yeast, the vacuoles typically occupy more than 70% of the cellvolume. Yeast vacuoles are related to lysosomes of animal cells, andcontain a variety of hydrolytic enzymes with an acidic pH in the lumen.

The sample is typically stained by passive means; that is the labelingsolution is combined with the sample being analyzed. The substrates ofthe present invention are introduced into the sample organelles byincubation of the sample in the labeling solution. Where the samplecontains a cell or cells, the cells are similarly stained by incubationof the cell or cells in the labeling solution. Alternatively, the sampleis stained by direct uptake of the substrate from a thin film of thesubstrate applied to a plate, microplate or cell well. Any other methodof introducing the substrates into the sample cell, such asmicroinjection of a labeling solution, can be used to accelerateintroduction of the substrates into the cellular cytoplasm. Typicallythe substrates will be introduced into the sample cell by incubation inthe labeling solution, or by microinjection. Preferably the substrate isintroduced to the sample by incubation in the labeling solution.Microinjection of substrate solution is used when labeling of the acidicorganelles in a single cell is desired, within a colony of other samplecells.

A number of reagents and conditions are known to affect the pH gradientof acidic organelles, and therefore the staining by the substrates ofthe invention, including but not limited to nutrients (for examplecarbohydrates such as glucose) and selected drugs.

The substrates of the present invention are generally non-toxic toliving cells and acidic organelles. Sample cells have been incubated in200 uM substrate solution for 72 hours without observable ill effects.Stained cells have been observed to undergo cell division, producingdaughter cells that also possess stained acidic organelles.

Optionally, the cells or isolated acidic organelles are washed toimprove the results of the staining procedure. Washing the sample cellor cells after incubation in the labeling solution, or optionally afterfixation or permeabilization, greatly improves the visualization of theacidic organelles. This is largely due to the decrease in non-specificbackground staining after washing. Satisfactory visualization of acidicorganelles is possible without washing by using low labelingconcentrations (for example <50 nM).

The sample can be observed immediately after staining of acidicorganelles becomes evident. After staining, the cells or isolated acidicorganelles in a sample are optionally fixed. Selected embodiments of thesubstrates described above are well retained in cells, and sample cellsstained with these substrates retain considerable visual staining afterfixation. A number of fixatives and fixation conditions are suitable forpracticing this invention. Useful fixatives include, but are not limitedto, formaldehyde, paraformaldehyde, formalin, glutaraldehyde, coldmethanol and 3:1 methanol acetic acid. Typically, cell fixation isaccomplished by incubating the stained cells in a 3.7% solution ofparaformaldehyde for about 15-30 minutes. Fixation is typically used topreserve cellular morphology and to reduce biohazards when working withpathogenic samples.

Fixation is optionally followed or accompanied by permeabilization, suchas with acetone, ethanol, DMSO or various detergents. Permeabilizationis utilized to allow bulky additional detection reagents to enter thecellular space that would ordinarily be impermeant to an intact cellularmembrane. A large variety of fixatives, fixation conditions, andpermeabilization agents are known in the art, and other methods offixing or permeabilizing sample cells in conjunction with the stains ofthe present invention will be obvious to one of ordinary skill.

The use of the acidic organelle substrates of the present invention isoptionally combined with the use of an additional detection reagent. Anadditional detection reagent is a reagent that produces a detectableresponse due to the presence of a specific cell component, intracellularsubstance, or cellular condition. One or more additional detectionreagents may be used in conjunction with the substrates of the presentinvention, before or after fixation and/or permeabilization. Theadditional detection reagent may be used to stain the entire cell, or acellular substructure. The visual signal of the acidic organellesubstrates of the present invention and the detectable response of theadditional detection reagent may be observed simultaneously orsequentially. The observation of acidic organelle staining and adetectable response that are spatially coincident indicate that theadditional detection reagent is associated with the acidic organelles. Avariety of measurements can be made within acidic organelles in thismanner, even when the additional detection reagent does not itselflocalize selectively within the acidic organelles.

One class of appropriate additional detection reagents are fluorescentnucleic acid stains. A wide variety of appropriate nucleic acid stainsare known in the art, including but not limited to, Thiazole Orange,ethidium homodimer, propidium iodide, Hoechst 33258, and DAPI.Additional useful nucleic acid stains are known to those of skill in theart. The use of an appropriate nucleic acid stain in conjunction withthe substrates of the present invention can be selected to allowsimultaneous observation of acidic organelles, nuclear DNA, cellular RNAand/or mitochondrial DNA. Of particular utility is an additionaldetection reagent that is a cell-permeant nucleic acid stain, allowingsimultaneous visualization of acidic organelles and the cell nucleus.

Other appropriate additional detection reagents include selectedfluorescent metal ion indicators described in U.S. Pat. No. 5,453,517 toKuhn et al. (1995), or U.S. Pat. No. 5,405,975 to Kuhn et al. (1995).

In another embodiment of the invention, an appropriate additionaldetection reagent is any probe that selectively stains a cellularorganelle such as the cell membrane, nucleus, Golgi apparatus,mitochondrion, endoplasmic reticulum, or is a second acidic organelleprobe.

Specific examples of additional detection reagents includemitochondria-targeted stains, such as Rhodamine 123. Additionalfluorescent stains specific for mitochondria are described in U.S. Pat.No. 5,459,268 to Haugland et al. (1995). The above mitochondrial stainsaccumulate in mitochondria, and are fixable therein, allowingsimultaneous visualization of both mitochondria and acidic organelles infixed and permeabilized cells.

In one embodiment, the additional detection reagent comprises: a) onemember of a specific binding pair or a series of specific binding pairs,and b) a means for producing a detectable response. A specific bindingpair member can be a ligand or a receptor. As used in this document, theterm ligand means any organic compound for which a receptor naturallyexists or can be prepared. A receptor is any compound or compositioncapable or recognizing a spatial or polar organization of a molecule,e.g. epitopic or determinant site. Ligands for which naturally occurringreceptors exist include natural and synthetic peptides and proteins,including avidin and streptavidin, antibodies, enzymes, and hormones;nucleotides and natural or synthetic oligonucleotides; lipids;polysaccharides and carbohydrates; lectins; and a variety of drugs,including therapeutic drugs and drugs of abuse and pesticides. Ligandsand receptors are complementary members of a specific binding pair, eachspecific binding pair member having an area on the surface or in acavity which specifically binds to and is complementary with aparticular spatial and polar organization of the other.

The additional detection reagent may be used in conjunction with enzymeconjugates to localize cellular receptors; to localize hybridizationprobes; or to probe cells and tissues that do not express the enzyme,for example, by enzyme-linked immunosorbent assay (ELISA), orenzyme-mediated histochemistry or cytochemistry, or otherenzyme-mediated techniques. Enzyme-mediated techniques take advantage ofthe attraction between specific binding pairs to detect a variety ofanalytes. In one embodiment, the additional detection reaction comprisesan enzyme substrate to produces a fluorescent precipitate in thepresence of the appropriate enzyme, as described in U.S. Pat. No.5,316,906 to Haugland et al. (1994) and U.S. Pat. No. 5,443,986 toHaugland et al. (1995).

In general, an enzyme-mediated technique uses an enzyme attached to onemember of a specific binding pair or series of specific binding pairs asa reagent to detect the complementary member of the pair or series ofpairs. In the simplest case, only the members of one specific bindingpair are used. One member of the specific binding pair is the analyte,i.e. the substance of analytical interest. An enzyme is attached to theother (complementary) member of the pair, forming a “complementaryconjugate”. Alternatively, multiple specific binding pairs may besequentially linked to the analyte, the complementary conjugate, or toboth, resulting in a series of specific binding pairs interposed betweenthe analyte and the detectable enzyme of the complementary conjugateincorporated in the specific binding complex.

The additional detection reagent also incorporates a means for producinga detectable response. A detectable response means a change in, oroccurrence of, a parameter in a test system which is capable of beingperceived, either by direct observation or instrumentally, and which isa function of the presence of a specifically targeted member of aspecific binding pair in a cell sample. Such detectable responsesinclude the change in, or appearance of, color, fluorescence,reflectance, pH, chemiluminescence, infrared emission, or the depositionof an electron-rich substrate. Appropriate labels to provide adetectable response include, but are not limited to, a visible orfluorescent dye, an enzyme substrate which produces a visible orfluorescent precipitate upon enzyme action (for example, the action ofhorseradish peroxidase upon diaminobenzidine), visible or fluorescentlabeled latex microparticles, or a signal that is released by the actionof light upon the reagent (e.g. a caged fluorophore that is activated byphotolysis, or the action of light upon diaminobenzidine).

At any time after or during staining, the sample is observed with ameans for detecting a visual signal present in the stained acidicorganelles. For example, when using a substrate of the present inventionthat produces a florescent visual signal, the sample is illuminated witha wavelength of light that results in a detectable fluorescenceresponse. In one embodiment of the invention, the fluorescently labeledorganelles are observed after the cell or cells have additionally beenfixed and/or permeabilized. Observation is accomplished using visiblelight microscopy, or alternatively, observation of the sample comprisesilluminating the stained sample with a wavelength of light appropriateto generate a fluorescent response, and visually examining the sample byuse of a microscope, or confocal microscope.

The sample is optionally illuminated at a wavelength of ultraviolet,visible or infrared light specific for optimal excitation of afluorophore present in the sample after enzyme action to remove theBLOCK group. Where the sample contains more than one BLOCK, or containsan additional detection reagent, illumination occurs at a wavelengththat generates a detectable fluorescence response in each fluorescentsubstrate or additional detection reagent, where said substrates anddetection reagents possess overlapping excitation maxima.

Typically, the substrates of the invention typically possess a strongabsorbance at visible wavelengths, typically at greater than 450 nm,preferably at greater than 500 nm, yet more preferably at greater than600 nm. The preferred substrates of the invention exhibit an extinctioncoefficient greater than 10,000/cm M, preferably at greater than30,000/cm·M. The fluorophores of the invention typically possess quantumyields of fluorescence emission that are greater than 0.3, preferablygreater than 0.7.

Optionally, the sample is observed using instrumentation. For example,where the sample contains a cell or cells, observation of the sample isaccomplished by illuminating the stained cell or cells with a wavelengthof light appropriate to generate a fluorescent response, andelectronically detecting and optionally quantifying the fluorescentemission of the stained acidic organelles using an appropriateinstrument, such as a fluorescence microscope equipped with a digitalcamera, fluorometer, fluorescent microplate reader, or a flow cytometer.

The observation of the fluorescent response of the sample optionallyincludes selecting or sorting the acidic organelles based upon theirfluorescent response. Typically the sample comprises cells havingstained acidic organdies, and the cells of the sample are sorted basedupon the staining of the individual cells. The step of sorting istypically accomplished using a flow cytometer or a fluorescencemicroscope.

The use of simple fluorescent dyes as sensitizing agents to enhancephotodynamic therapy (PDT) has been described by Boyer (U.S. Pat. No.5,189,029) and Morgan (U.S. Pat. No. 5,446,157). Photodynamic therapyrefers to the process wherein illumination is utilized to destroy cells,typically abnormal cells, that have previously been labeled with a dye.Several references, including Geze, et al. (Photochem. Photobiol. 20,23-35 (1993)) and Berg et al. (Int. J. Cancer 59, 814-822 (1994)) havepreviously indicated that the photolysis of dyes that are localized tolysosomes destroys tumor cells. Furthermore, the lysosomes of tumorcells are generally considered to have a lower pH than normal lysosomes(“Molecular Aspects of Anticancer Drug Action”, Neidel and Waring, Eds.,Macmillan, London; pp 233-286 (1983)). Selective uptake of PDT dyes intotumor cells in preference to normal cells is an important propertyallowing selective photodestruction of abnormal cells in the course ofPDT treatment, while minimizing the destruction of normal cells.

The method of the current invention has utility for photodynamictherapy, as described above, as the greater acidity of lysosomes intumor cells, will result in greater uptake of the acidotropic substratesin tumor cells. Photolysis of the stained cells will then result indestruction of the target tumor cells without affecting neighboringnormal cells and tissues. Although cells and tissues stained accordingto the present method are potential PDT targets, preferably the longwavelength fluorescent dyes used for PDT targeting of cells are thosethat absorb beyond 600 nm, more preferably those that absorb beyond 650nm, due to the enhanced penetration of light through tissue at thesewavelengths. Particularly preferred are the substrates of the inventionhaving fused aromatic substituents that are further substituted by aLINK-T moiety. Additional preferred substrates of the invention for PDTare those having bromine or iodine substituents.

Preferred substrate concentrations for labeling cells for PDT are thoseconcentrations that have been determined to produce the greatestselective uptake of substrate into abnormal cells without detriment tonormal cells, such that photolytic activity is maintained in theabnormal cells. As described above, micromolar concentrations ofsubstrate are effective in staining acidic organelles of live cells. Thesubstrates are applied to cells for PDT by means well understood in theart, including local or systemic injection, topical application,incorporation into liposomes or other means. substrate uptake into cellsis by passive diffusion or receptor-mediated uptake. Selectiveaccumulation in lysosomes is facilitated by the pH gradient that favorsuptake into the more acidic organelles. Photolysis is performed with anyexcitation source that is capable of producing light that can beabsorbed by the substrate, including lasers and light sources thatproduce infrared irradiation. This light may be delivered eitherdirectly to the cells that contain the substrate, or deliveredindirectly such as through an optical fiber. Fluorescence properties ofthe substrate can be used to guide and determine which cells are to beirradiated.

The examples below are given so as to illustrate the practice of thisinvention. They are not intended to limit or define the entire scope ofthis invention.

Example 1 Preparation of a Lysosomal β-Galactosidase Substrate withGreen Fluorescence after Enzyme Reaction

The following compound was prepared:

5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluorescein

To a dry 100 mL round bottom flask under anhydrous N_(2(g)) was addedN-hydroxysuccinimide (5.75 g, 50 mmol), dissolved in trifluoroaceticanhydride (20.0 mL, 144 mmol) and allowed to stir at room temperaturefor 1.5 h. This reaction mixture was rotary evaporated under reducedpressure and co-evaporated with dry toluene (3×20 mL) at 50° C. anddried in vacuo to give O-trifluoroacetyl-N-hydroxysuccinimide as a whiteamorphous crystalline solid (10.57 g, 100%).

Under anhydrous conditions, a sample ofO-trifluoroacetyl-N-hydroxysuccinimide (10.57 g, 50 mmol) was dissolvedin anhydrous DMF (20 mL) and 5(6)-carboxy-2′,7′-dichlorofluorescein(5.20 g, 12 mmol) and dry pyridine (10.0 mL, 130 mmol) were added. Thismixture was allowed to stir at room temperature under anhydrousconditions overnight. The reaction mixture was then poured intoice-water (300 mL) with stirring and extracted with ethylacetate (200mL). The aqueous layer was extracted again with ethylacetate (100 mL)and the combined ethylacetate layers were washed with water (200 mL) andbrine solution (200 mL), dried over anhydrous sodium sulfate, filtered,evaporated and dried in vacuo to give a bright orange amorphous solid(4.65 g, 84%). Chromatography (TLC: SiO₂ plate, 7:3ethylacetate:methanol irrigant, R_(f)=0.53 and 0.79) indicatedapproximately a 1:1 mixture of the two isomeric active esters.

A sample of the above NHS-esters (4.65 g, 10.1 mmol) was dissolved inanhydrous DMF (50 mL) and unsym-dimethylethylenediamine (1.65 mL, 15mmol) added. This mixture was allowed to stir under anhydrousconditions, at room temperature overnight. Ethylacetate (200 mL) wasadded with stirring for 30 min. and the pale orange precipitatefiltered, washed with ethylacetate and dried in vacuo to give the titledimethylaminoethyl amides (2.88 g, 57%). The combined ethylacetatefiltrates from above were extracted with 1N HCl/H2O solution (2×100 mL)and the resulting aqueous layers washed with dichloromethane (2×25 mL),evaporated to dryness and the resulting orange oil was triturated withdiethylether to give a second crop of the title compound (as theHCl-salt form) (2.20 g, total yield=97%).

5(6)-(2-dimethylaminoethyl)carboxamido)-2′,7′-dichlorofluorescein-3′,6′-di-O-β-D-galactopyanoside,octaacetate

A sample of5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluorescein (3.20g, 6.37 mmol) was suspended in anhydrous dichloromethane (40 mL), THF(40 mL) and anhydrous acetonitrile (20 mL). To this solution was addedacetobromogalactose (6.76 g, 16.4 mmol), dry silver carbonate (2.20 g,7.96 mmol) and sym-collidine (2.0 mL, 15.1 mmol), the flask covered inAl-foil (darkness) and allowed to stir under anhydrous N_(2(g)) for 72h. Additional acetobromogalactose (5.5 g) and silver carbonate (1.75 g)were added and left the reaction to continued stirring as above for 18h. The reaction mixture was filtered through a Celite™ pad and thesilver salts washed with excess dichloromethane. The filtrates werecombined and evaporated to a brown oil, redissolved in dichloromethane(100 mL) and washed with water (100 mL), 1 N HCl solution (100 mL)saturated sodium bicarbonate solution (2×100 mL) 1 N HCl solution (2×100mL) and brine (100 mL). The resulting dichloromethane layer was driedover anhydrous sodium sulfate, filtered and applied to a column ofsilicagel 60 (70-230 mesh, 100×45 mm) and eluted using a gradientelution method of dichloromethane:ethylacetate (0-20%). Fractionscontaining the second major product to elute from the column werecombined and evaporated to give an off-white foam (2.52 g, 34%).

5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluorescein-3′,6′-di-O-β-D-galactopyanoside

To a flame dried 250 mL one-neck, round-bottom flask under dry N_(2(g))was added5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluorescein-3′,6′-di-O-β-D-galactopyanoside,octaacetate (1.50 g, 3.0 mmol). The sample was suspended in anhydrousmethanol (100 mL), cooled to 0° C. (ice-bath) under an atmosphere of drynitrogen gas and 25% (w/v) sodium methoxide/methanol solution (1.0 mL,250 mg) added with stirring. The solution was allowed to react underanhydrous conditions for 3 hours at 0° C., warmed to room temperatureand neutralized with washed, dry IRC50 (H+) resin (2 grams). The resinwas filtered and washed with methanol, and the combined filtratesevaporated to dryness and dried in vacuo to an off white solid (1.05grams, 98%). Crystallization from methanol:diethylether (1:10, 2×) gavea product homogeneous by TLC analysis (7:3 ethylacetate:methanolirrigant; Rf=0.08).

Example 2

Preparation of an Esterase Substrate with Blue Fluorescence Emissionupon Enzyme Activity.

The following compound was prepared:

To a flame dried 50 mL round-bottom flask under an atmosphere of dryN₂(g) was weighed3-(2′-dimethylaminoethylcarboxamidomethyl)-6-chloro-7-hydroxycoumarin(M1247, 99 mg, 0.3 mmole). This sample was suspended in anhydrousdichloromethane (20 mL), cooled to 0° C. (ice-bath) and acetic anhydride(1.0 mL, 10.6 mmole) and dry pyridine (1.0 mL, 12.4 mmole) added. Thereaction was allowed to stir at 0° C. for 2 hours and atroom-temperature overnight. The reaction mixture was diluted withdichloromethane (30 mL) and poured into ice-water (100 mL) withstirring. The organic layer was separated and washed with saturatedsodium bicarbonate solution (1×100 mL) and brine solution (1×100 mL),dried over anhydrous sodium sulfate, filtered, evaporated andco-evaporated with dry toluene (2×10 mL) to give a clear glass (50 mg,42%) homogeneous by TLC analysis (irrigant 9:1 dichloromethane:methanol;Rf=0.85).

Example 3 Preparation of a Lipase Substrate with Green Fluorescence UponEnzyme Activity

The following compound was prepared:

5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluorescein,di-O-octanoate

5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluorescein (152mg, 0.295 mmole) was suspended in anhydrous dichloromethane (10 mL) andcooled to 0° C. (ice-bath) while under anhydrous conditions under drynitrogen gas. To this solution was added octanoyl chloride (250 uL, 1.62mmole) and dry pyridine (1120 uL, 14.42 mmole) and the reaction mixturewas placed in an ultrasonic bath for 30 min. to complete dissolution ofthe starting materials. This reaction mixture was allowed to react atambient temperature under anhydrous conditions overnight. The reactionwas diluted with dichloromethane (50 mL) and poured into ice-water (50mL), extracted, and the resulting organic layer washed with ice-water(1×50 mL), saturated sodium bicarbonate solution (1×50 mL) 1 N HClsolution (1×50 mL) and brine solution (1×50 mL), dried over anhydroussodium sulfate, filtered, evaporated and dried in vacuo to give a cleartan oil (184 mg, 81%). TLC analysis showed two spots of essentiallyequal intensity for the two isomeric 5(6)-derivatives at Rf=0.42 and0.48 (irrigant=9:1 CH₂Cl₂:MeOH). ¹H-NMR (d₆-DMSO) (mixture of 5 and6-isomers) δ: 9.1 (m, 0.5H); 9.0 (m, 0.5H); 8.5 (s, 0.5H); 8.2 (m, 0.5);8.1 (m, 0.5H); 8.0 (d, 0.5H); 7.8 (s, 0.5H); 7.6 (m, 0.5H); 7.6 (s, 1H);7.5 (d, 0.5H); 7.4 (d, 0.5H); 7.2 (s, 0.5H); 7.1 (s, 0.5; H); 3.7 (m,1H), 3.6 (m, 1H); 3.3 (m, 1H), 3.2 (m, 1H); 2.4 (2, 6H); 1.6 (m, 4H);1.3 (br. s, 20H); 1.8 (t, 6H).

Example 4 Preparation of a β-Glucosidase Substrate with BlueFluorescence upon Enzyme Activity

The following compound was prepared:

3-Acetoxyethyl-6-chloro-7-hydroxycoumarin

To a flame-dried 50 mL round-bottom flask is weighed 4-chlororesorcinol(1.44 g, 10 mmole) and diethyl-1,3-acetonedicarboxylate (1.82 mL, 10mmole) and trifluoroacetic acid (10.0 mL) added. This mixture is heatedto reflux (98° C.) in an oil-bath for 18 hours, cooled toroom-temperature and poured into ice-water (100 mL) with stirring for 1hour. The resulting off-white precipitate is filtered and washed withice-cold water, dried in air and in vacuo overnight to give a yellowcrystalline powder (1.29 g, 4.56 mmole, 46%) TLC (irrigant=7:3ethylacetate:methanol, Rf=0.86).

3-(2′-dimethylaminoethylcarboxamidomethyl)-6-chloro-7-hydroxycoumarin

To a dry 50 mL round-bottom flask was weighed3-Acetoxyethyl-6-chloro-7-hydroxycoumarin (500 mg, 1.77 mmole) andunsym-dimethylethylenediamine (5 mL, 45.38 mmole) added. This mixturewas heated with stirring to reflux for 2 hours and at room-temperatureovernight, evaporated and co-evaporated with dry toluene (4×25 mL) anddried in vacuo overnight. The resulting reddish oil was trituratedrepeatedly with anhydrous diethylether to give a tan powder (0.58 g,100%) homogeneous by TLC analysis (1:1 ethylacetate:methanol) (Rf=0.10).¹H-NMR (d₆-DMSO) δ: 8.2 (br. s, 1H, NH); 7.8 (d, 1H); 6.6 (d, 1H); 6.0(d, 1H); 3.5 (m, 2H); 3.1 (m, 2H); 2.1 (s, 6H).

7-O-(2,3,4,6-tetra-O-acetyl□β-D-glucopyranosyl)-6-chloro-3-(2′-dimethylaminoethylcarboxamidomeihyl)coumarin

Under anhydrous conditions,3-(2′-dimethylaminoethylcarboxamidomethyl)-6-chloro-7-hydroxycoumarin(354 mg, 1.09 mmole) was suspended in anhydrous dichloromethane (20 mL)containing 3A molecular sieve (0.5 g) and anhydrous acetonitrile (10mL), acetobromoglucose (686 mg, 1.66 mmole), sym-collidine (500 uL, 3.78mmole) and dry silver carbonate (247 mg, 0.9 mmole) added. This mixturewas allowed to react under anhydrous nitrogen in the dark for 72 hoursat room temperature. The reaction mixture was filtered through a Celite™pad and the greenish precipitate washed with excess dichloromethane. Thecombined filtrates were extracted with brine solution (2×100 mL),saturated sodium bicarbonate solution (1×100 mL), 0.2 N sodiumthiosulfate solution (1×100 mL) and brine solution (1×100 mL), driedover anhydrous sodium sulfate, filtered and evaporated to dryness. Theproduct was purified on a 20×20 cm preparative TLC plate (1 mmthickness) with elution using 8:2 dichloromethane:acetonitrile aseluent. The highest Rf band was removed from the plate, eluted with 1:1dichloromethane:acetonitrile as solvent, filtered and dried in vacuo togive the title compound (126 mg) homogeneous by TLC analysis.

7-O-β-D-glucopyranosyl-6-chloro-3-(2′-dimethylaminoethylcarboxamidomethyl)coumarin

A sample of7-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-6-chloro-3-(2%dimethylaminoethylcarboxamidomethyl)coumarin (113 mg, 0.17 mmole) wassuspended in anhydrous methanol (15 mL) under dry nitrogen gas, and 25%sodium methoxide in methanol solution (800 uL, 200 mg NaOMe) added. Thismixture was allowed to stir under anhydrous conditions for 6 hours,neutralized with washed, dry IRC50 (H+) resin (0.5 g) and filtered. Theresin was washed with fresh methanol and the combined filtratesevaporated and dried in vacuo overnight to a clear oil which wastriturated with anhydrous diethylether (25 mg) to give a white solid (58mg, 69%). TLC (7:3 ethylacetate:methanol irrigant showed one spot,Rf=0.75). ¹H-NMR (d₆-DMSO) δ: 7.8 (s, 1H); 7.5 (s, 1H); 6.3 (s, 1H); 5.2(d, 1H, H-1); 4.9-4.0 (m, 4H, —OH); 3.6-2.8 (m, 10H, CHO ring andtargeting arm protons); 2.4 (s, 611, —CH3's).

Example 5 Preparation of an Arylsulfatase Substrate with GreenFluorescence Emission Upon Enzyme Activity

The following compound was prepared:

5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluoresceindisulfate, disodium salt

A solution of5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluorescein (66 mg,0.128 mmoles) was dissolved in anhydrous pyridine (5.0 mL) underanhydrous N_(2(g)), cooled to 0° C. (ice-bath) and chlorosulfonic acid(43 mg, 0.64 mmole) added dropwise with stirring. This solution wasallowed to react at 0° C. for 2 hours and at room-temperature overnight.The solution was evaporated under reduced pressure (T<40° C.) andco-evaporated with dry toluene (2×30 mL), dried briefly in-vacuo andtreated with 0.1 mM NaOH solution (6.4 mL) and the resulting solutionapplied to a column of Sephadex LH-20 resin, with elution using water.Fractions containing the quenching non-fluorescent product were combinedand lyophilized to give a light tan solid (46 mg, 50%). TLC(irrigant=9:1:1 dichloromethane:methanol:acetic acid) Rf=0.0 originallyquenching, but fluorescent upon H+ treatment.

Example 6 Preparation of an Acid Phosphatase Substrate with GreenFluorescence Upon Enzyme Activity

The following compound was prepared:

(m, 0.5H); 7.5 (s, 0.5H); 7.2 (d, 0.511); 6.8 (s, 1H); 6.4 (s, 1H); 3.2(m, 2H); 3.1 (m, 2H); 2.5 (s, 6H, —CH3's); 2.1 (s, 6H, —OAc's).

Example 8 Preparation of an Esterase Substrate Containing a MorpholinoTargeting Group with Green Fluorescence after Enzyme Activity

The following compound was prepared:

5(6)-(3-N-morpholinopropyl)carboxamido)-2′7′-dichlorofluorescein

O-trifluoroacetyl-N-hydroxysuccinimide (11.18 g, 53 mmol) was dissolvedin anhydrous dimethylformamide (20 mL) and5(6)-carboxy-2′,7′-dichlorofluorescein (5.20 g, 12.0 mmole) added, withstirring. This mixture was allowed to stir at room temperature underanhydrous conditions for 12 hours after which time, TLC analysis(mini-workup with ethylacetate:water; ethylacetate layer; irrigant=7:3ethylacetate:water) indicated that the reaction was complete. Thereaction mixture was then poured into ice-water (300 mL) with stirringand extracted with ethylacetate (200 mL). The layers were separated andthe aqueous layer washed again with ethylacetate (100 mL). The combinedethylacetate layers were washed with water (200 mL),

Preparation of an Esterase Substrate with Green Fluorescence afterEnzyme Activity.

The following compound was prepared:

5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluoresceindiacetate

A suspension of5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluorescein (101mg, 0.20 mmole) in anhydrous dichloromethane (20 mL) was cooled to 0° C.(ice-bath) and acetic anhydride (1.0 mL, 10.6 mole) and dry pyridine(1.0 mL, 12.4 mmole) was added. This mixture was allowed to stir at 0°C. for two hours and at ambient temperature overnight. The reactionmixture was diluted with dichloromethane (100 mL) poured into ice-water(100 mL), extracted and the organic layer washed with ice-cold saturatedsodium bicarbonate solution (100 mL), water (100 mL) and brine solution(200 mL). The resulting organic layer was dried over anhydrous sodiumsulfate filtered, evaporated and dried in vacuo to give a pale tan solidthat was triturated with diethyl ether, centrifuged, decanted and theoff-white crystals dried in vacuo (40 mg, 33%). TLC analysis (9:1CH₂Cl₂:MeOH) showed a two spots Rf=0.58 and 0.42) ¹H-NMR (d₆-DMSO) (5and 6 isomers) δ: 8.7 (t, 0.5H); 8.6 (t, 0.5H); 8.3 (s, 0.5H); 8.0 (d,0.5H); 7.9 brine solution (200 mL), 1 N HCl/H₂O (200 mL) and brine (100mL). A significant amount of orange precipitate formed which wasfiltered and dried (1.79 g, appears to be a single isomer, by TLCanalysis (7:3 ethylacetate:methanol; Rf=0.5). The filtrate was driedover anhydrous sodium sulfate, filtered, evaporated and dried in vacuoto give a bright orange solid (TLC analysis (7:3 ethylacetate:methanolirrigant), 2 isomers, Rf=0.53 and 0.79) (4.65 g, 6.44 g total yield70%). A sample of the mixed isomeric NHS esters (1.00 g, 2.17 mmole) wasdissolved in anhydrous DMF, and 3-aminopropyl-N-morpholine (1.3 mL, 8.90mmole, 4.1 equiv.) added with stirring. This mixture was allowed to stirunder anhydrous conditions overnight, evaporated under vacuum to abright red oil (1.53 g) that was triturated with ethyl acetate (50 mL)(30 min.) and diethyl ether (50 mL) (overnight) to remove excessmorpholino compound. The product was filtered and redissolved in drymethanol, evaporated and dried overnight in vacuo to give an orangesolid (542 mg, 44%) homogeneous by TLC analysis (irrigant=7:3ethylacetate:methanol) Rf=0.06).

5(6)-(3-N-morpholinopropyl)carboxamido)-2′7′-dichlorofluoresceindiacetate

The sample of5(6)-(3-N-morpholinopropyl)carboxamido)-2′7′-dichlorofluorescein (542mg, 0.95 mmole) was dissolved in anhydrous dichloromethane (10 mL) andacetic anhydride (1.0 mL, 10.6 mmole) and dry pyridine (1.0 mL, 12.4mmole) added. This reaction mixture was allowed to stir overnight underanhydrous conditions. The reaction mixture was then diluted withdichloromethane (50 mL) and poured into ice-water (150 mL) withstirring. After stirring for 30 min. to destroy excess acetic anhydride,the layers were separated and the organic layer was with ice-coldsaturated sodium bicarbonate solution (25 mL), 1 N HCl solution (25 mL)and water (25 mL). The organic layer was dried over anhydrous sodiumsulfate, evaporated and dried in vacuo to a clear, pale tan oil,homogeneous by TLC analysis (irrigant=9:1 dichloromethane:methanol;Rf=0.58 and 0.55) as two closely separated isomers, quenching at UV 254nm, but non-fluorescent. A biocompatible staining solution was preparedby dissolving 86 mg of the above compound in anhydrous DMSO (1.312 mL)to give a 100 mM solution.

Example 9 Preparation of a β-Galactosidase Substrate with GreenFluorescence after Enzyme Activity and a Morpholino Targeting Group

The following compound was prepared:

5(6)-(3-N-morpholinopropyl)-carboxamido)-2′7′-dichlorofluoresceindi-β-D-Galactopyranoside, octaacetate

A sample of5(6)-(3-N-morpholinopropyl)-carboxamido)-2′7′-dichlorofluorescein (1.60g, 2.80 mmole) was dried in vacuo overnight, placed under anhydrousN_(2(g)), and acetobromogalactose (2.90 g, 7.0 mmole, 2.5 equiv.) added.These solids were suspended in anhydrous dichloromethane (15 mL) andanhydrous acetonitrile (15 mL) and solid, dry silver carbonate (970 mg,3.5 mmole), sym-collidine (930 uL, 7.0 mmole), dry 3 A molecular sieves(1 g) were added. This reaction mixture was covered in Al-foil(darkness) and allowed to stir under anhydrous conditions for 3 days.After this time, the reaction mixture was filtered through a bed ofdiatomaceous earth (Celite™ 545) and the precipitate washed with excessdichloromethane. The combined filtrates were washed with water,saturated aqueous sodium bicarbonate solution, 1 N HCl, 0.2 N sodiumthiosulfate solution and water (each 1×100 mL). The organic layer wasdried over anhydrous sodium sulfate, filtered, evaporated to a lowvolume and applied to a slurry-packed column of silicagel 60 (70-230mesh, 125 g, 40×160 mm) prepared in dichloromethane. The product waseluted by gradient elution, using dichloromethane (500 mL), 8:2dichlorormethane:ethylacetate (1.5 L), 7:3 dichloromethane:ethylacetate(500 mL), 6:4 dichloromethane:ethylacetate (500 mL) and 9:1dichloromethane:methanol (500 mL). Fractions containing the first majorquenching (UV 254 nm) product to elute from the column were combined,evaporated and dried in vacuo to give a clear oil (540 mg, 16%). TLC(8:2 dichloromethane:ethylacetate, Rf=0.13). ¹H-NMR (d₆-DMSO) (mixtureof 5 and 6 isomers) δ: 8.4 (d, 0.5H); 8.3 (m, 0.5H); 8.2 (s, 0.5H); 7.8(s, 0.511); 7.6 (d, 0.5H); 7.2-6.9 (m, 4.511); 6.1 (d, 2H, H-1); 5.4-5.0(m, 6H); 4.5 (m, 2H); 4.2-3.9 (8H); 2.4 (s, 4H); 2.1-1.9 (4s, 24H); 1.2(t, 2H).

5(6)-(3-N-morpholinopropyl)-carboxamido)-2′7′-dichlorofluoresceindi-β-D-Galactopyranoside

A sample of5(6)-(3-N-morpholinopropyl)-carboxamido)-2′7′-dichlorofluoresceindi-β-D-galactopyranoside, octaacetate (540 mg, 0.44 mmole) was dried invacuo overnight, placed under anhydrous N_(2(g)) and dissolved inanhydrous methanol (40 mL). To this solution was added 25% (w/v) sodiummethoxide in methanol (90 mg, 1.66 mmole) and this mixture allowed tostir under anhydrous conditions for 2.5 hours. The reaction mixture wasthen neutralized with washed, dry IRC-50 (H+) resin, allowing the resinto stir for about 30 min. The resin was filtered and washed with excessdry methanol, evaporated to a low volume (about 5 mL) (rotaryevaporator, T<35° C.) and crystallized by adding dry diethylether (100mL). The solution was stored at 4° C. overnight to completecrystallization, and the off-white crystals filtered and washed withfresh diethylether to give an off-white crystalline solid (190 mg, 48%).TLC analysis (irrigant=9:1:1 dichloromethane:methanol:acetic acid,Rf=0.05). The solid was very hygroscopic and difficult to weigh.Biocompatible solutions of the product were prepared in DMSO for cellstaining experiments. ¹H-NMR (d₆-DMSO) (5 and 6 isomers) δ: 8.4 (d,0.5H); 8.3 (m, 0.5H); 8.2 (d, 0.5H); 7.8 (s, 0.5H); 7.5 (d, 0.5H); 7.2(s, 2H); 7.0 (s, 0.5H); 6.9 (s, 2H); 6.5 (d, 2H); 6.1 (d, 2H, H-1); 5.1(m, 2H, H-4); 5.2-4.2 (m, 10H); 3.9-3.2 (20H); 2.5 (t, 4H); 1.0 (t, 2H).

Example 10 Preparation of an Esterase Substrate with Red Fluorescenceafter Enzyme Activity

The following compound was prepared:

5(6)-Carbaxynaphthofluorescein

To a dry 300 mL round-bottom flask was added trimellitic anhydride (9.60g, 50 mmole), 1,6-dihydroxynaphthalene (16.02 grams, 100 mmole) andfused zinc chloride (11.60 grams, 85.1 mmole). These solids were mixedmanually and heated to 180° C. (oil-bath) neat, overnight. The reactionwas cooled to room-temperature and the purple solid digested with 4 NNaOH solution (150 mL), filtered to remove zinc salts and theprecipitate washed with water. The filtrate was cooled to 0° C. (addedDI-ice) and neutralized with concentrated HCl (˜30 mL) to pH 3. Theresulting dark red sample was stored at 4° C. overnight to completecrystallization, filtered and the dark red solid washed with water untilthe filtrate was neutral. The solid was dried in air and in vacuoovernight to give a dark red powder (43 g). Repeated attempts torecrystallize the sample were unsuccessful. An 8.0 gram sample of theabove crude product was dissolved in methanol (50 mL) containingtriethylamine (TEA, 1 mL), and adsorbed to Celite™ 545 (about 10 grams),evaporated and dried in vacuo. This sample was applied to a slurrypacked column of silicagel 60 (70-230 mesh) (250 grams, 45×300 mm) andeluted by gradient elution with 9:1 dichloromethane:methanol (1.5 L),9:1 dichloromethane:methanol (1.5 L) containing 1% TEA (300 mL), 9:1dichloromethane:methanol (1.5 L) containing 2% TEA, 8:2dichloromethane:methanol (1.5 L) containing 2% TEA (2 L), and 7:3:1dichloromethane:ethylacetate:methanol with 2% TEA (500 mL). Fractionscontaining the second major product (blue) to elute from the column werecombined and evaporated to give a dark blue solid (4.23 g). This samplewas redissolved in water (200 mL), cooled to 0° C. (ice-bath) andacidified with concentrated HCl (˜20 mL) to give a dark red precipitatethat was filtered and washed with water until the filtrate was neutral.The red solid was dried in air and in vacuo to give 3.13 grams (TLCanalysis, 7:3:1 dichloromethane:ethylacetate:methanol+2% TEA) Rf=0.13).¹H-NMR (d₆-DMSO) δ: 8.7 (d, 2H); 8.5 (s, 1H); 8.2 (m, 2H); 7.7 (s, 1H);7.4 (dd, 2H); 7.3 (dd, 2H); 7.2 (d, 2H); 6.7 (dd, 2H).

5(6)-(2-dimethylaminoethyl)carboxamido)-naphthofluorescein

A sample of 5(6)-carboxynaphthofluorescein (470 mg, 0.95 mmole) wasdissolved in dry DMF (10 mL) and O-trifluoroacetyl-N-hydroxysuccinimide(665 mg, 3.15 mmole) and dry pyridine (800 uL, 9.93 mmoles) were added.This solution was allowed to stir at room temperature under anhydrousconditions for 18 hours. The reaction mixture was then diluted withethylacetate (100 mL) and extracted with ice-water (2×100 mL), 1 N HCl(1×100 mL) and brine solution (1×100 mL). The combined aqueous layerswere back-extracted with ethylacetate (25 mL) and the combined organiclayers were dried over anhydrous sodium sulfate, filtered and evaporatedin vacuo to give a blue solid (180 mg, 32%). TLC analysis (irrigant=9:1dichloromethane:methanol; Rf=0.30). A sample of the NHS esters (150 mg,0.262 mmole) was dissolved in anhydrous DMF (10 mL) andunsym-dimethylethylenediamine (50 uL, 453 mmole) was added. Thissolution was allowed to stir under dry N_(2(g)) at room temperatureovernight. The reaction was evaporated and dried in vacuo overnight to atan oil, that was resuspended in acetonitrile (50 mL) and water (10 mL).The solution was acidified with 1 N HCl until the color changed fromyellow to red. The sample was evaporated and co-evaporated withacetonitrile (2×20 mL), and crystallized from cold acetonitrile (10 mL)to give a purple solid (43 mg, 29%) TLC analysis (irrigant=5:5:3:1pyridine:ethylacetate:acetic acid:water) Rf=0.42). ¹H-NMR (d₆-DMSO)exhibited two isomers in approx. 2:1 ratio.

5(6)-(2-dimethylaminoethyl)carboxamido)-naphthofluorescein diacetate

A sample of5(6)-(2-dimethylaminoethyl)carboxamido)-2′7′-dichlorofluorescein (21 mg,35 umole) was suspended in dry dichloromethane (2 mL), cooled to 0° C.(ice-bath) under dry N_(2(g)) and acetic anhydride (0.5 mL, 5.3 mmole)and dry pyridine (0.5 mL, 6.2 mmole) were added. This reaction mixturewas allowed to stir under anhydrous conditions at 0° C. for 2 hours andat room temperature overnight. The resulting clear, pale orange solutionwas treated with ice-cold water (2 mL), and extracted. The resultingorganic layer was washed with ice-cold saturated sodium bicarbonatesolution (3 mL), 0.1 N citric acid solution (2 mL) and water (2 mL). Theresulting organic layer was dried over anhydrous sodium sulfate,filtered, evaporated and dried in vacuo to give a light tan sample (16mg) that was recrystallized from methanol:water (1:1, 10 mL). Theresulting light salmon colored powder was dried in vacuo (yield=14 mg,64%).

Example 11 Preparation of a Benzoxazolylcoumarin Esterase Substrate withBlue Fluorescence after Enzyme Activity

The following compound was prepared:

2-Cyanoacetimide, Ethyl ester, hydrochloride

A solution of diethylether (30 mL) and absolute ethanol (9.01 mL, 154.4mmole) was cooled to 0° C. (ice-bath) and acetyl chloride (5.49 mL,77.20 mmole) added slowly with stirring. This solution was allowed toreact for 30 minutes, and added to a solution of malononitrile (5.10grams, 77.20 mmole) in anhydrous diethylether (20 mL). The above mixturewas allowed to react at 0° C. overnight. The abundant white precipitatewas filtered, dried in air and in vacuo to give a light yellowcrystalline product after drying (7.88 g, 69%). TLC analysis (7:3ethylacetate:methanol) showed a single product Rf=0.77).

4-Amino-3-hydroxybenzoic acid, ethyl ester, hydrochloride salt

To a dry 100 mL round-bottom flask was added absolute ethanol (50 mL)and the solution cooled to 0° C. (ice-bath) under dry N_(2(g)). Acetylchloride (5.0 mL) was added slowly with stirring and this solutionallowed to stir under anhydrous conditions for 2 hours to prepareanhydrous 1.40 M HCl in ethanol. To this solution was added4-amino-3-hydroxybenzoic acid (2.50 g, 16.32 mmole) and the mixtureallowed to stir as above at room temperature overnight. TLC(irrigant=7:1:1:1 ethylacetate:methanol:water:acetic acid, Rf=0.85)indicated complete conversion to the ethyl ester. Yield 3.5 g (99%).

5-Carboxyethyl-2-cyanomethylbenzoxazole

A solution of 2-cyanoacetimide, ethyl ester, hydrochloride (1.49 g, 10mmole) and 4-amino-3-hydroxybenzoic acid, ethyl ester, hydrochloridesalt (2.17 g, 10 mmole) in anhydrous dichloromethane (25 mL) was heatedto gently reflux overnight, cooled to room temperature, diluted withdichloromethane (100 mL) and extracted successively with water, 1 N HCl,saturated sodium bicarbonate solution and water (1×100 mL each). Theresulting organic layer was dried over anhydrous sodium sulfate,filtered, evaporated and dried in vacuo to give an oil (0.98 g, 43%)that was used without further purification. TLC analysis (9:1dichloromethane:methanol) Rf=0.88).

3-(3′-Carboxyethylbenzoxazolyl)-7-hydroxycoumarin

5-Carboxyethyl-2-cyanomethylbenzoxazole (0.98 g, 4.26 mmole) wasdissolved in absolute ethanol (20 mL) and 2,4-dihydroxybenzaldehyde (589mg, 4.26 mmole) and ammonium acetate (406 mg) added. The solution wasallowed to stir under anhydrous conditions overnight. The reaction wascooled to −18° C., and the abundant yellow-orange precipitate wasfiltered and washed with minimum ice-cold absolute ethanol, dried in airand in vacuo to give the title compound (900 mg, 60%) as a bright yellowsolid. TLC analysis (irrigant=9:1 dichloromethane:methanol) Rf=0.65. Asecond crop (240 mg) was obtained from the filtrate with concentrationand cooling, but was the free acid (TLC irrigant=9:1:1dichloromethane:methanol:acetic acid, Rf=0.25).

3-(3′-[Carboxamidoethyl-2-dimethylamino]benzoxazolyl)-7-hydroxycoumarin

A sample of 3-(3′-Carboxyethylbenzoxazolyl)-7-hydroxycoumarin (400 mg,1.14 mmole) was suspended in unsym-dimethylethylenediamine (4.0 mL, 36.3mmole), the vial capped and allowed to stir at room temperature for 72hours. The reaction mixture was poured into diethylether (200 mL) tocrystallize, and after drying gave the title compound as a tan powder(72 mg, 16%). TLC analysis (irrigant=9:1:1dichloromethane:methanol:acetic acid) Rf=0.02.

3-(3′-[Carboxamidoethyl-2-dimethylamino]benzoxazolyl)-7-acetoxycountarin

A sample of3-(3′-[Carboxamidoethyl-2-dimethylamino]benzoxazolyl)-7-hydroxycoumarin(72 mg, 0.183 mmoles) was suspended in dry dichloromethane (2 mL) usingultrasonication, and acetic anhydride (1.0 mL, 10.6 mmole) and drypyridine (1.0 mL, 12.4 mmole) added. This mixture was allowed to stirovernight, and the resulting product solution, diluted withdichloromethane (30 mL) and extracted with water (1×50 mL), saturatedsodium bicarbonate solution (1×50 mL), 1 N HCl (1×50 mL) and water (1×50mL). The resulting dichloromethane layer was dried over anhydrous sodiumsulfate, filtered and evaporated. Drying in vacuo provided an off-whitesolid (66 mg, 83%). A biocompatible solution of this substrate wasprepared by dissolving 44 mg in dry DMSO (1.0 mL) for staining livecells for esterase activity.

Example 12 Preparation of a Hexosaminidase Substrate with RedFluorescence after Enzyme Activity

The following compound was prepared:

4-Chloro-2-nitrosoresorcinol

A solution of anhydrous ethanol (100 mL) under dry nitrogen gas wascooled in an ice-bath (0° C.) and solid sodium metal (2.3 g, 100 mmole)added with stirring until dissolved. 4-chlororesorcinol (14.4 g, 100mmole) was added with stirring until dissolved (20 min.) and a solutionof N-butylnitrite (10.3 g, 100 mmole) in absolute ethanol (10 mL) addeddropwise with stirring. This solution was allowed to react for 3 hoursat (0° C.), and then poured into ice-water (300 mL) and acidified with 1N aqueous HCL solution (˜100 mL) until the pH was 2-3. The resultingsolid was collected by filtration, washed with water and dried in vacuoto give a solid (6.4 g, 37%). TLC analysis showed a single spot(irrigant=5:1 ethylacetate:methanol) Rf-0.40.

3-Chloro-5-carboxyresorufin

4-Chloro-2-nitrosoresorcinol (6.10 g, 35.1 mmole) and3,5-dihydroxybenzoic acid (5.41 g, 3.51 mmole) was suspended inanhydrous methanol (140 mL) and cooled to −5° C. in an ice-methanolbath. Solid manganese dioxide (3.40 g) was added followed by conc.sulfuric acid (3.7 mL) dropwise keeping the temperature between 0° and5° C. The ice-methanol bath was removed and the dark red mixture allowedto stir at room temperature for 2 hours. The solution was then filteredthrough a fluted filter paper, and conc. ammonium hydroxide added untilthe filtrate changed to a dark green-blue color. This solution was againfiltered through a Celite™ 545 pad and additional ammonium hydroxideadded (20 mL). This basic solution was cooled in an ice-bath withstirring and the pH was adjusted to 2 with aqueous HCl solution. Theresulting solution was evaporated to dryness and redissolved in methanol(30 mL) and applied to silicagel 60, with evaporation. The solid samplewas applied to a column of silicagel 60 (70-230 mesh, 45×2 cm) andeluted by gradient elution with 20%, 25% and 30% methanol indichloromethane (1 L each) and 40% methanol in dichloromethane (500 mL).The third set of fractions contained the title dye (2.70 g, 26%). TLC(irrigant=1:1 dichloromethane:methanol, Rf=0.58).

3-Chloro-5-carboxyresorufin, ethyl ester

To a dry flask under dry nitrogen gas was added absolute ethanol (50 mL)and cooled to in an ice-bath. Acetyl chloride (3.56 mL, to make 1 MHCl/EtOH) was added and this solution allowed to stir at 0° C. for 15min. A sample of 3-Chloro-5-carboxyresorufin (303 mg, 1.04 mmole) wasadded and the solution allowed to stir under anhydrous conditions atroom temperature overnight. The solvents were evaporated at reducedpressure (rotovap) and co-evaporated with abs. ethanol (2×10 mL) anddried in vacuo to give a dark red solid (0.36 grams) that was purifiedby column chromatography (silicagel 60, 70-230 mesh column 25×400 mm,elution with 20:1 dichloromethane:methanol). Fractions containing thepure ethyl ester were combined and evaporated to give a red solid (41mg, 12%). TLC analysis (9:1 dichloromethane:methanol) Rf=0.39.

3-Chloro-5-(3-N-morpholinopropyl)carboxamidoresorufin

A sample of 3-Chloro-5-carboxyresorufin, ethyl ester (41 mg, 0.13 mmole)was dissolved in N-(3-aminopropyl)-morpholine (410 uL, 2.8 mmole) andallowed to stir at room temperature for 72 hours. TLC analysis(irrigant=7:3 ethylacetate:methanol) indicated that all of the startingmaterial was consumed and a new product (Rf=0.45) was formed. Thereaction mixture was evaporated to dryness and coevaporated with drymethanol. The final oily product was triturated with diethylether togive a red solid (55 mg, 96%).

3-Chloro-5-(3-N-morpholinopropyl)carboxamidoresorufin2,3,6-tri-O-acetyl-2-deoxy-2-N-Acetyl-β-D-Galactopyranoside

A solution of 3-chloro-5-(3-N-morpholinopropyl)carboxamidoresorufin (73mg, 0.174 mmole), acetobromo-N-acetylgalactosamine (86 mg, 0.20 mmole)and silver carbonate (28 mg, 0.105 mmole) was prepared in anhydrousdichloromethane, and placed under an atmosphere of dry nitrogen gas. 3Amolecular sieves (0.4 g) and 2,4,6-collidine (28 uL, 0.20 mmole) wereadded and the flask covered in Al-foil (darkness) and allowed to stirunder anhydrous conditions for 3 days. The resulting solution wasdiluted with dichloromethane (50 mL) and filtered through a Celite™ pad,and the filtrate was extracted with water (50 mL), satd. sodiumbicarbonate solution (50 mL), water (50 mL), 1 N aqueous HCl (50 mL),0.2; N sodium thiosulfate solution (50 mL) and water (50 mL). The finalorganic layer was dried over anhydrous sodium sulfate, filtered andevaporated to an orange solid (120 mg). TLC analysis exhibited a singlemajor product (irrigant=25:1 dichloromethane; triethylamine) Rf=0.44contaminated with residual sugar (Rf=0.95) upon sulfuric acid charring.This sample was used for synthesis of the title N-acetylgalactoside.

3-Chloro-5-(3-N-morpholinopropyl)carboxamidoresorufin2-deoxy-2-N-Acetyl-β-D-Galactopyranoside

Under anhydrous conditions, the crude sample of3-Chloro-5-(3-N-morpholinopropyl)carboxamidoresorufin2,3,6-tri-O-acetyl-2-deoxy-2-N-Acetyl-b-D-Galactopyranoside (30 mg) wasdissolved in anhydrous methanol (20 mL) under an atmosphere of drynitrogen gas and 25% (w/v) sodium methoxide in methanol (670 uL, 84 mgNaOMe) added with stirring. This solution was allowed to stir underanhydrous conditions for 4 hours, and neutralized with washed, dry IRC50(H+) resin (about 3 grams) until neutral. The resin was filtered andwashed with dry methanol and the combined filtrates were evaporated anddried in vacuo to give an orange glass (20 mg) homogeneous by TLCanalysis (irrigant=7:3 ethylacetate:methanol) Rf=0.1. A biocompatiblestaining solution was prepared by dissolving this sample in dry DMSO anddetermining the concentration by measuring the absorbance at 457 nmusing an extinction coefficient of 17.9K.

Example 13 Preparation of an Alternate Targeting Group with a Long ChainAlkyl Group for Increased Lipophilicity

The following compound was prepared:

N—BOC—N′-methylethylenediamine

A solution of N-Methylethylenediamine (2.00 g, 27.0 mmol) in DMF/water(1:1, 30 mL) was adjusted to ph 7 with 6 N HCl. A solution ofDi-t-butyldicarbonate (5.89 g, 27 mmol) in DMF (20 mL) was added to thestirred solution at room temperature. The pH of the reaction mixture wasmaintained between 6 and 7 by the periodic addition of 1 N NaOH. Thereaction was stirred until the pH remained constant. The reaction wasdiluted with water (150 mL) and the resulting solution was washed withethylacetate (3×100 mL). The pH was adjusted to ˜11.5 with 10 N NaOH andthe resulting solution was extracted with ethylacetate (3×200 mL). Theextract was filtered through a cotton plug and the solvent was removed.The resulting liquid was stirred in vacuo overnight at room temperatureto yield N—BOC—N′-methylethylene-diamine andN—BOC—N-methylethylenediamine as a 1:1 mixture (1.73 g, 41% massrecovery). ¹H NMR (CDCl₃) δ: 1.43 and 1.45 (9H), 1.78 (s, 2H), 2.45 (s,1.5H), 2.73 (t, 1H), 2.82 (t, 1H), 2.87 (s, 1.51H), 3.27 (m, 2H).

N—BOC—N′-decyl-N′-methylethylenedianzine

To a stirred solution of N—BOC—N′-methylethylenediamine andN—BOC—N-methylethylenediamine (1:1, 1.73 g, 4.96 mmol each) in MeOH (50mL) were added decanal (9.31 g, 59.6 mmol) followed by sodiumcyanoborohydride (585 mg, 9.31 mmol) at room temperature. The reactionwas stirred overnight at room temperature. The reaction was concentratedon a rotevaporator and the concentrate was diluted with water (50 mL).The pH was lowered to ˜2 with 6 N HCl and the acidic solution wasextracted with ethylacetate (3×100 mL). The amine products wereextracted into the organic phase. The ethylacetate portion was washedwith 0.1 N NaOH (1×100 mL) and brine (2×100 mL) and the solvent wasremoved. The resulting liquid was dried overnight in vacuo at roomtemperature. The product was isolated by column chromatography on silicagel column (4×15 cm). Side products were eluted from the column with 1and 2% MeOH in CH₂Cl₂ then the product was eluted with 5 and 10% MeOH inCH₂Cl₂. The solvent was removed from the product fractions and theresidue was dried in vacuo at it to yieldN—BOC—N′-decyl-N′-methylethylenediamine as a pale yellow solid (470 mg,21%): TLC (silica gel, 1 MeOH:9 CH₂Cl₂) R_(f)=0.6; ¹H NMR (DMSO-d₆) δ0.82 (t, 3H), 1.23 (s, 16H), 1.35 (s, 9H), 2.10 (s, 3H), 2.25 (m, 4H),2.95 (m, 2H), 6.80 (s, 1H).

N-decyl-N-methylethylenediamine-bis-trifluoroacetic acid salt

A solution of N—BOC—N′-decyl-N′-methylethylenediamine (450 mg, 1.43mmol) in TFA (15 mL) was allowed to stand at room temperature for 30min. The TFA was removed on a rotevaporator and the resulting oil wasdried in vacuo for 1 h to give a semi-solid. The semi-solid wastriturated with ether (15 mL) and the resulting suspension was storedovernight in a freezer. The resulting solid was collect by vacuumfiltration and was washed repeatedly with ether. The filter cake wasdried in vacuo to yieldN-decyl-N-methylethylenediamine-bis-trifluoroacetic acid salt as acolorless crystalline solid (470 mg, 74%): ¹H NMR (DMSO-d₆) δ 1.80 (t,3H), 1.22 (s, 16H), 1.58 (m, 2H), 2.80 (s, 3H), 3.08 (m, 2H), 3.25 (m,2H), 8.22 (bs, 3H).

Example 14 Preparation of an Alternate Targeting Group with Two LongChain Alkyl Groups for Increased Lipophilicity

The following compound was prepared:

N—BOC—N′,N′-Diheptylethylenediamine

To a stirred solution of N—BOC-ethylenediamine (500 mg, 3.12 mmol) inMeOH (15 mL) were added heptanal (802 mg, 7.02 mmol) followed by sodiumcyanoborohydride (588 mg, 9.36 mmol) and the reaction was stirredovernight at room temperature. The reaction was concentrated on arotevaporator and the concentrate was diluted with water (50 mL). The pHwas lowered to ˜2 with 6 N HCl and the acidic solution was extractedwith ethylacetate (3×100 mL). The layer was washed with 0.1; N NaOH(1×100 mL) and brine (2×100 mL) and the solvent was removed. The productwas isolated by column chromatography on silica gel. The solvent wasremoved from the product fractions and the residue was dried in vacuo atroom temperature to yield two major productsN—BOC—N′,N′-diheptylethylenediamine (0.51 g, 46%): TLC (silica gel, 1MeOH:9 CH₂Cl₂) R_(f)=0.81; and N—BOC—N′-heptylethylenediamine (0.23 g,15%): TLC (silicagel 9:1 dichloromethane:methanol) Rf=0.66.

N,N-Diheptylethylenediamine-bis-trifluroacetic acid salt

A solution of N—BOC—N′,N′-diheptylethylenediamine (x mg, x mmol) in TFA(x mL) was allowed to stand at room temperature for 30 min. The TFA wasremoved on a rotevaporator and the residue was dried in vacuo for 1 h atroom temperature. The residue was triturated with ether (x mL) and theresulting suspension was stored overnight in a freezer. The resultingsolid was collect by vacuum filtration and was washed repeatedly withether. The filter cake was dried in vacuo at room temperature to yieldN,N-diheptylethylenediamine, bis-trifluoroacetic acid salt as a solid(613 mg, 90%). Deblocking of the mono-heptyl targeting group wasperformed in a similar manner to yield the N-heptylethylenediamine,bis-trifluoroacetic acid salt.

Example 15 Preparation of an Esterase Substrate with Blue Fluorescenceafter Enzyme Activity and Containing an Increased Lipophilic TargetingGroup

The following compound was prepared:

2-(6-Chloro-7-hydroxycoumarin-4-yl)-N-[2-(N-decyl-N-methyl-amino)ethyl]acetamide

A solution of 1 N KOH (7.34 mL) was added to a suspension of Ethyl6-Chloro-7-hydroxycoumarin-4-carboxylic acid (1.00 g, 3.54 mmol) inethanol (75 mL) with stirring at room temperature to give a yellow tobrown solution. The ethanol portion was concentrated. The concentrateand the oily residue were dissolved in H2O (50 mL) and the pH of theresulting solution was adjusted to ˜6 to 7 with 1 N HCl. The solutionwas washed with ethylacetate (3×100 mL) to removed any residual ethylester. The pH was lowered to ˜2 with 1 N HCl and the acidic solution wasextracted with ethylacetate (3×100 mL). The extract was washed withsaturated brine (2×100 mL) and was dried over Na₂SO₄. Solvent removalgave the carboxylic acid as a beige solid (670 mg, 75%): TLC (silica gel1:3 MeOH:EtOAc) R_(f)=0.2. Without purification the above acid (670 mg,2.63 mmol) was dissolved in DMF (25 mL) and the resulting solution wasadded to solid N-hydroxysuccinimide trifluoroacetate (3.74 g, 17.7mmol). Pyridine (5 mL) was added to the resulting solution and thereaction was stirrer overnight at room temperature. The reaction waspoured onto ice and diluted with H₂O (250 mL). The pH was lowered to 2with 1 N HCl and the solution was extracted with ethylacetate (2×100mL). The extract was washed with saturated brine (2×100 mL) and wasdried over Na₂SO₄. The solvent was removed and the gummy residue waswashed with a small amount of ethylacetate. The resulting solid wascollected by vacuum filtration and was dried in vacuo to yield theN-hydroxy-succinimidyl ester as a beige solid (750 mg, 81%): TLC (silicagel 1:9 MeOH:CH₂Cl₂) R_(f)=0.8. A portion of the aboveN-hydroxysuccinimidyl ester (280 mg, 0.796 mmol) was combined withN-decyl-N-methylethylenediamine-bis-trifluoroacetic acid salt (282 mg,0.637 mmol) and Ethyl diisopropyl amine (169 mg, 1.31 mmol) in DMF (1mL). The resulting solution was stirred overnight at room temperature.Trifluoroacetic acid (114 mg, 1.00 mmol) was added to the reaction. Thevolatile portion was removed in vacuo at 50° C. to give a gummy solid.The solid was triturated with ethylacetate (1 to 2 mL) to give asuspension. The solid was collected by vacuum filtration and was washedwith ethylacetate. The filter cake was dried in vacuo to yield2-(6-Chloro-7-hydroxycoumarin-4-yl)-N-[2-(N-decyl-N-methyl-amino)ethyl]acetamideas a colorless solid (148 mg, 51%): TLC (silica gel 1:9 MeOH:CH₂Cl₂)R_(f)=0.3; ¹H NMR (DMSO-d₆) δ: □0.81 (t, 3H), 1.22 (s, 16H), 1.58 (m,2H), 2.60 (s, 3H), 2.80-3.20 (m, 4H), 3.70 (s, 2H), 6.23 (s, 1H), 6.90(s, 1H), 7.78 (s, 1H), 8.43 (t, 1H).

2-(6-Chloro-7-acetoxycoumarin-4-yl)-N-[2-(N-decyl-N-methyl-amino)ethyl]acetamide

A sample of this2-(6-Chloro-7-hydroxycoumarin-4-yl)-N-[2-(N-decyl-N-methyl-amino)ethyl]acetamide(26 mg, 0.06 mmole) was dissolved in anhydrous dichloromethane (5 mL)and acetic anhydride (0.5 mL, 5.3 mmole) and dry pyridine (0.5 mL, 6.2mmole) added. This mixture was allowed to stir overnight, and theresulting solution, diluted with dichloromethane (30 mL) and extractedwith water (1×25 mL), saturated sodium bicarbonate solution (1×25 mL), 1N HCl (1×25 mL) and water (1×25 mL). The resulting dichloromethane layerwas dried over anhydrous sodium sulfate, filtered and evaporated. Dryingin vacuo provided an off-white solid (22 mg, 79%). A biocompatiblesolution of this substrate was prepared by dissolving 22 mg in dry DMSO(0.446 mL) for staining live cells for esterase activity.

Example 16 Preparation of a β-Glucosidase Substrate with BlueFluorescence after Enzyme Activity and an Increased Lipophilic TargetingGroup

The following compound is prepared:

A sample of2-(6-Chloro-7-hydroxycoumarin-4-yl)-N-[2-(N-decyl-N-methyl-amino)ethyl]acetamide(50 mg, 0.11 mmole) was dried in vacuo overnight, placed under anhydrousN2(g), and acetobromoglucose (77 mg, 0.28 mmole, 2.5 equiv.) added.These solids were suspended in anhydrous dichloromethane (15 mL) andsolid, dry silver carbonate (39 mg, 0.14 mmole), sym-collidine (37 uL,0.28 mmole), dry 3 A molecular sieves (0.5 g) were added. This reactionmixture was covered in Al-foil (darkness) and allowed to stir underanhydrous conditions for 3 days. After this time, the reaction mixturewas filtered through a bed of diatomaceous earth (Celite™ 545) and theprecipitate washed with excess dichloromethane. The combined filtrateswere washed with water, saturated aqueous sodium bicarbonate solution, 1N HCl, 0.2; N sodium thiosulfate solution and water (each 1×50 mL). Theorganic layer was dried over anhydrous sodium sulfate, filtered,evaporated to a low volume and applied to a slurry-packed column ofsilicagel 60 (70-230 mesh, 50 g, 40×60 mm) prepared in dichloromethane.The product was eluted by gradient elution, using dichloromethane (500mL), 8:2 dichlorormethane:ethylacetate (500 mL), 6:4dichloromethane:ethylacetate (500 mL) and 9:1 dichloromethane:methanol(500 mL). Fractions containing the first major quenching (UV 254 nm)product to elute from the column were combined, evaporated and dried invacuo to give a clear oil (70 mg). TLC (8:2dichloromethane:ethylacetate, Rf=0.75).

This peracetate was dried in vacuo overnight, placed under anhydrousN_(2(g)) and dissolved in anhydrous methanol (40 mL). To this solutionwas added 25% (w/v) sodium methoxide in methanol (90 mg, 1.66 mmole) andthis mixture allowed to stir under anhydrous conditions for 2.5 hours.The reaction mixture was then neutralized with washed, dry IRC-50 (H+)resin, allowing the resin to stir for about 30 min. The resin wasfiltered and washed with excess dry methanol, evaporated to a low volume(about 5 mL) (rotary evaporator, T<35° C.) and crystallized by addingdry diethylether (100 mL). The solution was stored at 4° C. overnight tocomplete crystallization, and the off-white crystals filtered and washedwith fresh diethylether to give an off-white crystalline solid (41 mg,55%). TLC analysis (irrigant=9:1:1 dichloromethane:methanol:acetic acid,Rf=0.22). Biocompatible solutions of the product were prepared in DMSOfor cell staining experiments.

Example 17 Preparation of a β-Galactosidase Substrate with BlueFluorescence after Enzyme Activity and an Increased Lipophilic TargetingGroup

The following compound is prepared:

A sample of2-(6-Chloro-7-hydroxycoumarin-4-yl)-N-[2-(N-decyl-N-methyl-amino)ethyl]acetamide(50 mg, 0.11 mmole) was dried in vacuo overnight, placed under anhydrousN2(g), and acetobromogalactose (77 mg, 0.28 mmole, 2.5 equiv.) added.These solids were suspended in anhydrous dichloromethane (15 mL) andsolid, dry silver carbonate (39 mg, 0.14 mmole), sym-collidine (37 uL,0.28 mmole), dry 3 A molecular sieves (0.5 g) were added. This reactionmixture was covered in Al-foil (darkness) and allowed to stir underanhydrous conditions for 3 days. After this time, the reaction mixturewas filtered through a bed of diatomaceous earth (Celite™ 545) and theprecipitate washed with excess dichloromethane. The combined filtrateswere washed with water, saturated aqueous sodium bicarbonate solution, 1N HCl, 0.2 N sodium thiosulfate solution and water (each 1×50 mL). Theorganic layer was dried over anhydrous sodium sulfate, filtered,evaporated to a low volume and applied to a slurry-packed column ofsilicagel 60 (70-230 mesh, 50 g, 40×60 mm) prepared in dichloromethane.The product was eluted by gradient elution, using dichloromethane (500mL), 8:2 dichlorormethane:ethylacetate (500 mL), 6:4dichloromethane:ethylacetate (500 mL) and 9:1 dichloromethane:methanol(500 mL). Fractions containing the first major quenching (UV 254 nm)product to elute from the column were combined, evaporated and dried invacuo to give a clear oil (66 mg). TLC (8:2dichloromethane:ethylacetate, Rf=0.7).

This peracetate was dried in vacuo overnight, placed under anhydrousN_(2(g)) and dissolved in anhydrous methanol (40 mL). To this solutionwas added 25% (w/v) sodium methoxide in methanol (90 mg, 1.66 mmole) andthis mixture allowed to stir under anhydrous conditions for 2.5 hours.The reaction mixture was then neutralized with washed, dry IRC-50 (H+)resin, allowing the resin to stir for about 30 min. The resin wasfiltered and washed with excess dry methanol, evaporated to a low volume(about 5 mL) (rotary evaporator, T<35° C.) and crystallized by addingdry diethylether (100 mL). The solution was stored at 4° C. overnight tocomplete crystallization, and the off-white crystals filtered and washedwith fresh diethylether to give an off-white crystalline solid (29 mg,38%). TLC analysis (irrigant=9:1:1 dichloromethane:methanol:acetic acid,Rf=0.20). Biocompatible solutions of the product were prepared in DMSOfor cell staining experiments.

Example 18 Preparation of an L-Alanyl Peptidase Substrate with GreenFluorescence after Enzyme Reaction

The following compound was prepared:

5(6)-Carboxyrhodamine 110

Under anhydrous conditions a mixture of trimellitic anhydride (4.80 g,25 mmole) and 3-aminophenol (5.46 g, 50 mmole) in anhydrousmethanesulfonic acid (15 mL, 231 mmole) was heated to 182° C. for 24hours with stirring. The reaction was cooled to room temperature andpoured into ice-water (200 mL) and allowed to stir for 1 hour tocrystallize. The resulting red solid was filtered, and washed withwater, dried in air and in vacuo briefly (1 hour) and then digested with2 N NaOH solution (40 mL). The resulting sodium salt was cooled to 0° C.(added ice-cubes directly to the solution) and concentrated HCl (12 M,10 mL) was added to acidify (pH 3). The resulting red precipitate wasfiltered and washed with water until the filtrate was neutral, dried inair and in vacuo to give a red solid 6.72 g (72%), homogeneous by TLCanalysis (irrigant=9:1:1 dichloromethane:methanol:acetic acid; Rf=0.02).¹H NMR (d₆-DMSO) (mixture of 5 and 6 isomers) δ: 8.5 (d, 0.5H); 8.3 (dd,0.5H); 8.25 (m, 1.5H); 8.2 (br. s, 3H, —NH); 7.9 (s, 0.5H); 7.6 (d,0.5H); 7.0 (d, 2H); 6.8 (m, 3.5H).

N,N′-di-FMOC-L-Alanyl-5(6)carboxyrhodamine 110

Under anhydrous conditions,N-alpha-(9-fluorenylmethyloxycarbonyl)-L-Alanine (FMOC-L-Ala-OH, 847 mg,2.72 mmole) was dissolved in anhydrous THF (5 mL) and cooled in amethanol/ice bath (−5° C.). N-methylmorpholine (329 uL, 3.0 mmole) andisobutylchloroformate (392 uL, 3.0 mmole) were added with stirring, andthis mixture was allowed to stir for 30 min. at −5° C., and then warmedto room temperature. To this suspension was added a solution of5(6)-carboxyrhodamine 110 (112 mg, 0.272 mmole) in dry DMF (0.5 mL)containing N-methylmorpholine (33 uL, 0.3 mmole) along with two DMFrinses (1 mL each). This solution was allowed to stir at roomtemperature overnight with stirring. The reaction mixture was added towater (50 mL) and extracted with ethylacetate (3×25 mL). The resultingcombined ethylacetate layers were washed with water (25 mL), saturatedsodium carbonate solution (2×25 mL), water (25 mL) and brine solution(25 mL). The final ethylacetate layer was dried over anhydrous sodiumsulfate, filtered and dried to an orange foam that was redissolved indichloromethane (3 mL) and purified on a column of silicagel 60 (70-230mesh, 19×2.5 cm) slurry packed in 3:1 hexanes:ethylacetate. The productwas eluted by gradient elution using 25% ethylacetate:hexanes (300 mL),30% ethylacetate:hexanes (400 mL) and 40% ethylacetate:hexanes (600 mL).Fractions containing the product were combined and evaporated and driedin vacuo to give a colorless solid (211 mg). ¹H NMR (CDCl₃) wasconsistent with the title structure, with some extra peaks from someexcess free amino acid. This product was used for the synthesis of theamide.

N,N′-di-L-Alanyl-5(6)-(2-dimethylaminoethyl)carboxamido)-rhodamine 110

The crude sample of N,N′-di-FMOC-L-Alanyl-5(6)carboxyrhodamine 110 (211mg, 0.205 mmole) was dissolved in dry DMF under anhydrous N_(2(g)) andethyl 3-(dimethylamino)propyl carbodiimide (43 mg, 0.225 mmole, 1.1equiv.) and N-hydroxysuccinimide (NHS, 76 mg, 0.225 mmole, 1.1equivalents) added. This solution was allowed to stir at roomtemperature for 5 hours, and unsym-dimethylethylenediamine (225 uL, 2.0mmole, 10 equiv.) was added and the reaction continued stirring for 18hours. After this time, ethylacetate (25 mL) and water (25 mL) wereadded, the layers were separated, and the ethylacetate layer was washedwith water (25 mL), saturated sodium bicarbonate solution (25 mL) andwater (25 mL). The organic layer was dried over anhydrous sodiumsulfate, filtered, and the solvent removed by vacuum distillation(rotovap). The resulting NHS ester was purified by preparative TLC(20×20 cm, 1 mm thickness), using 30% ethylacetate:dichloromethane aseluent, to give an off-white solid (168 mg), showing two main productsby TLC analysis (irrigant=3:1 dichloromethane:methanol). The sample wasredissolved in dry DMF (5 mL) and piperidine (200 uL, 2.0 mmole) added.After stirring for 6 hours at room temperature, the solution was driedin vacuo and triturated with diethylether (2×25 mL) to give a whitepowder (111 mg, 92%).

Example 19 Preparation of Cells in Culture for Labeling

Human skin fibroblasts from Lysosomal Storage Disease patients (Krabbe,Tay-Sachs, Sandhoff, Wolman, and Gaucher diseases) were obtained fromthe Istituto Giannina Gaslini (Genova, Italy). Cell lines weremaintained in RPMI 1640 Medium (HyClone) supplemented with 9% FetalBovine Serum (Gibco) and 1X Antibiotic/Antimycotic (Gibco). Cells weregrown to 90% confluence and passaged by splitting at a 1:5 ratio. Cellswere incubated at 37° C., with 5% CO₂ atmosphere.

Human skin fibroblasts from a healthy specimen were obtained from theCoriell Institute for Medical Research (Camden, N.J.). Cells weremaintained in Minimum Essential Medium Eagle (EMEM) (Lonza) supplementedwith 9% Fetal Bovine Serum (Gibco) and 1× Antibiotic/Antimycotic(Gibco). Cells were grown to 90% confluence and passaged by splitting ata 1:5 ratio. Cells were incubated at 37° C., with 5% CO₂ atmosphere.

NIH 3T3 and CRE BAG 2 (murine tumor fibroblast) cell lines were obtainedfrom the American Type Culture Collection (Manassas, Va.). Cells weremaintained in Dulbecco's Modified Eagles Medium (DMEM) (Sigma)supplemented with 9% Fetal Bovine Serum (Gibco) and 1×Antibiotic/Antimycotic (Gibco). Cells were grown to 70% confluence andpassaged by splitting at a 1:10 ratio. Cells were incubated at 37° C.,with 5% CO₂ atmosphere.

Example 20 Preparation of Labeling Solutions for Mammalian Cell Systems

The desired substrate of the invention is separately dissolved in DMSOto prepare a 10 mM stock solution. The stock solution is kept sealed insmall aliquots, at −20.degree. C. The stock solution is kept frozen atall times until use, and exposure to light is minimized. One aliquot ofdye stock is taken from the freezer immediately before an experiment andthawed completely at room temperature. The labeling solution is thenprepared by adding the dye stock solution to fresh serum-free culturemedium (37.degree. C.) in an amount sufficient to make final dyeconcentrations ranging from 1-200 μM. Dye stock solutions are added suchthat the final concentration of DMSO in the labeling solution does notexceed 2%.

Example 21 Labeling of Lysosomes and Acidic Organelles in Live AnimalCells

Cells prepared according to Example 19 are transferred to the labelingsolution containing either M1268, M1299, M1322, or M1344, and incubatedat 37.degree. C. for 15 to 150 minutes. The cells are then washed withpre-warmed fresh medium that does not contain serum (37° C.) andobserved using a fluorescence microscope equipped with appropriatefilters, such as an XF68 filter set (Omega Optical).

Example 22 Staining of Lysosomes and Chromatin in Living Cells Using anAdditional Detection Reagent

Cells prepared according to Example 19 are transferred to the labelingsolution containing 10 μM M1344 and 2 μg/mL of the nuclear stain DAPIand incubated at 37° C. for 90 minutes. The cells are then washed withfresh, pre-warmed culture medium that does not contain serum andexamined under a fluorescence microscope equipped with multibandpassfilter set, such as an XF68 filter set (Omega Optical). As both dyes areorganelle-specific, the lysosomes and other acidic organelles stain arestained a bright fluorescent green, while the nuclei are simultaneouslystained fluorescent blue.

Example 23 Differential Staining with for Lipase Activity with LipaseInhibition

Cells were cultured as described in Example 19. Medium was removed fromhealthy exponentially growing human skin fibroblasts and cells werewashed with Phosphate Buffered Saline to remove residual Fetal BovineSerum. Tetrahydrolipostatin (THL) inhibitor solution was prepared bydiluting 10 mM stock solution (in 6:4 DMSO:EtOH) to 200 μM inunsupplemented EMEM. Control solution was prepared by addition of 6:4DMSO:EtOH at equal concentration as in inhibitor solution. Cells wereincubated in either control or inhibitor solution for 3 hours at 37° C.,5% CO₂ atmosphere.

Control or inhibitor solutions were removed and cells were washed withPhosphate Buffered Saline. Substrate solution was prepared by diluting 5mM stock solution (in DMSO) to 10 μM in unsupplemented EMEM. Cells wereincubated in substrate solution for 1.5 hours at 37° C., 5% CO₂atmosphere. Substrate solution was then removed, and cells washed 3times with Phosphate Buffered Saline. Cells were imaged using an XF68filter set (Omega Optical). Four images of each treatment were processedusing cell profiler imaging software to produce a mean objectfluorescence intensity for each group.

The following experimental conditions were used. Cells were incubated inserum-free EMEM containing 200 μM, 750 μM, or 1 mM Tetrahydrolipstatin.Significantly lower staining of lysosomes was observed in inhibitedcells versus control cells as shown in Table 12.

TABLE 12 [THL] MEAN OBJECT INTENSITY  1 MM 0.113 750 UM 0.127 200 UM0.171 UNTREATED 0.209

Example 24

Healthy skin fibroblast cells GM03440 were seeded into a 6-well tissueculture plate (FALCON 353046) and cultured in Minimum Essential MediumEagle (EMEM) supplemented with 9% Fetal Bovine Serum (Gibco) and 1XAntibiotic/Antimycotic solution (Gibco). Cells were allowed to adhere tothe plate surface. Cells were incubated for 48 hours, 37° C., 5% CO₂humidification. Medium was removed, and cells washed with PhosphateBuffered Saline to remove residual serum and treatment compounds. Cellswere incubated in serum-free EMEM containing 20 μM M1322 for 1.5 hours.Medium was removed, and cells were then washed three times withPhosphate Buffered Saline and mounted in the same. Cells were imagedusing an XF06 (DAPI) filter set (Omega Optical). Nine images of eachtreatment (Chloroquine, Retinoic Acid, Colchicine, Untreated) wereprocessed using Cell Profiler imaging software to produce a mean objectfluorescence intensity for each group.

The following experimental conditions were used. Chloroquine (10 mM DMSOstock) was added to a final concentration of 10 μM. Retinoic Acid (10 mMEtOH stock) was added to a final concentration of 10 μM. Colchicine (1mM stock in sterile Phosphate Buffered Saline) was added to a finalconcentration of 1 μM. One set of wells was left untreated. The results,shown in Table 2, indicate that the effect of drug administration onesterase enzyme activity in these cell lines could be monitored usingthe substrate M1322 in a live cell format.

TABLE 2 MEAN OBJECT PUBLISHED EFFECT TREATMENT INTENSITY ON LYSOSOMESUNTREATED 0.136 N/A RETINOIC ACID 0.149 INDUCER COLCHICINE 0.181INCREASES PH CHLOROQUINE 0.161 INCREASED STORAGE OF POLAR LIPIDS.

Example 25 Staining of Mammalian Cells for Aryl Sulfatase Activity

Cells were cultured as described in Example 19. Medium was removed fromhealthy exponentially growing human skin fibroblasts and cells werewashed with Phosphate Buffered Saline to remove residual Fetal BovineSerum. Substrate solution was prepared by diluting 10 mM stock solution(in DMSO) to 200 μM in unsupplemented EMEM. Cells were incubated insubstrate solution for 1.5 hours at 37° C., 5% CO₂ atmosphere. Substratesolution was then removed, and cells washed 3 times with PhosphateBuffered Saline. Cells were imaged using a B-2A filter set (Nikon).

Imaged cells showed punctuate fluorescent green staining. Very littlecytosolic staining of the cells was observed.

Example 26 Detection of an Additional Detection Reagent in Lysosomes

GM03440 cells are incubated for 30 minutes in a solution of culturemedium and 100 pg/mL Datura stramonium (jimson weed) lectin conjugatedto fluorescein at 37° C. in a tissue culture incubator. This greenfluorescent lectin has been shown to be taken up by live cells andtrafficked to lysosomes. The cells are rinsed twice in fresh culturemedium at 37° C., and then are allowed to recover in culture mediumwithout lectin for 4 hours at 37° C. Following recovery, the cells areincubated with 20 μM M1322 and diluted in growth medium for 30 minutesat 37° C. The cells are then rinsed in growth medium without substrateand mounted in the same.

Observation of the stained sample reveals blue and blue-green structureswithin the cells. The observed blue-green fluorescence indicatescolocalization of the green fluorescent lectin conjugate with the blueemission of the substrate turnover in lysosomes. The blue structuresindicate the staining of acidic organelles that are not lysosomes.

Example 27 The Effect of NH₃ Alkalization on Acidic Organelle Stainingin Live Cells

Cell samples prepared according to Example 19 are transferred to twotissue culture dishes. The cells in the first dish are pre-incubatedwith 1 mM NH₄OH for 30 minutes at 37.degree. C. The cells are thenwashed with fresh medium. The cells in both dishes are then incubatedwith a labeling solution that is 20 μM M1299 (as described in Example 2)at 37° C. for 30 minutes. The cells are then washed with pre-warmedfresh medium and observed using a fluorescence microscope equipped withan appropriate filter set.

The cells that were not initially alkalinized display good staining,with all of the lysosomes and other acidic organelles exhibiting abright green fluorescence. The initially alkalinized cells, however,show only weak staining of acidic organelles, displaying less than 10%of the fluorescence intensity of the control cells.

Example 28 Triple Labeling of Lysosomes, Golgi Apparatus and Nuclei inLiving Cells

A cell sample prepared according to Example 19 are transferred to atissue culture dish and incubated at 37.degree. C. for 30 minutes in alabeling solution that is 20 μM in M1299 (as described in Example 2),100 nM in BODIPY TR labeled ceramide (Molecular Probes, Inc., Eugene,Oreg.) and 30 nM in Hoechst 33258 (Molecular Probes, Inc., Eugene,Oreg.). The cells are then washed with fresh, pre-warmed culture mediumand examined under a fluorescence microscope equipped with anappropriate filter set, such as Omega XF68. The stained cells displaygreen fluorescent acidic organelles, red fluorescent Golgi apparatus,and blue fluorescent nuclei.

Example 29 Analysis of Cell Viability/Cytotoxicity

A cell sample prepared according to Example 19 is incubated at 37° C.for 30 minutes in a labeling solution that is 20 μM in M1299 and 50 nMin propidium iodide. The stained cells are then washed with fresh,pre-warmed culture medium. The cells are examined under a fluorescencemicroscope equipped with an appropriate filter set. Dead cells exhibitfluorescent red nuclei, while live cells exhibit green fluorescentacidic organelles. Cells that have damaged (i.e. permeant) cellmembranes, yet retain an acidic pH gradient with their acidic organelleswill display both green and red fluorescence.

1-40. (canceled)
 41. A substrate for detecting native enzyme activity inacidic organelles comprising the formula:T-LINK-F(R)-BLOCK(R′) where; T represents a targeting group that is aweakly basic amine containing compound that partitions the substrate tothe acidic organelle; F represents a reporter moiety comprising one ormore substituents R independently selected from the group consisting ofhydrogen, halogen, alkyl, alkenyl, aryl, and heteroaryl; BLOCK is amonovalent moiety adapted to be cleaved from the remainder of thesubstrate by action of a specific enzyme native to said acidicorganelle, resulting in a visible signal at the site of enzyme reaction;R′ is selected from the group consisting of an unsubstituted carboxylicacid ester and an alkyloxy substituted carboxylic acid ester; LINK hasthe formula —(CH2)a(CONH(CH2)b)z-, where a is an integer 0-5, b is 1-5and z is 0or
 1. 42. The substrate of claim 41, wherein said reportermoiety is selected from the group consisting of a fluorescent,chemiluminescent and chromogenic stain.
 43. The substrate of claim 41,wherein said BLOCK is selected from the group consisting of: (i) amonovalent moiety derived by removal of a hydroxy group from phosphateor sulfate, (ii) a biologically compatible salt of (i); (iii) amonovalent moiety derived by removal of a hydroxy group from a carboxygroup of an aliphatic, aromatic or amino acid or of a peptide; and (iv)a monovalent moiety derived by removal of an anomeric hydroxy group froma mono- or polysaccharide.
 44. The substrate of claim 41, wherein saidBLOCK further comprises one or more substituents (R′) that improvemembrane permeability of the substrate through cellular membranes. 45.The substrate of claim 41, wherein F is selected from the groupconsisting of an anthracene, a benzphenalenone, a coumarin, afluorescein, a naphthofluorescein, a naphthalene, a phenalenone, apyrene, a resorufin, a dioxetane, an indole, a luminol and a rhodamine.46. The substrate of claim 41, wherein T has the formula—CR_(c)R_(d)—NR_(e)R_(f), where: R_(c) and R_(d) are independentlyselected from the group consisting of hydrogen and an alkyl having 1-16carbons; R_(e) is alkyl and R_(f) is an alkyl group having 1-16 carbonatoms or N, Re and R_(f) form a nitrogen heterocyclic ring systemselected from the group consisting of morpholine, piperidine,pyrrolidine, piperazine, imidazole, oxazepine, azepine, pyrrole and analkyl substituted heterocyclic system with the alkyl having 1-18carbons.
 47. The substrate of claim 46, wherein amine substituents R_(e)and R_(f) form a nitrogen heterocycle selected from the group consistingof pyrrolidine, piperidine, piperazine, morpholine, imidazole, azepineand oxazepine.
 48. The substrate of claim 41, wherein T has the formula—CR_(c)R_(d)—NR_(e)R_(f), where: a) R_(c) and R_(d) are independentlyselected from the group consisting of hydrogen and an alkyl having 1-16carbons; b) R_(e) and R_(f) are alkyl groups.
 49. The substrate of claim48, where said alkyl groups R_(e) and R_(f) are substituted by a groupselected from the group consisting of halogen, carboxamide, oxy,hydroxy, mercapto and cyano.
 50. The substrate of claim 48, whereinamine substituents R_(e) and R_(f) are independently an alkyl grouphaving 1-6 carbons.
 51. The substrate of claim 41, wherein saidsubstrate has the formula:


52. The substrate of claim 41, wherein said substrate has the formula:


53. The substrate of claim 41, wherein said substrate has the formula:


54. The substrate of claim 41, wherein said substrate has the formula:


55. The substrate of claim 41, wherein said substrate has the formula:


56. The substrate of claim 41, wherein said substrate has the formula:


57. The substrate of claim 41, wherein said substrate has the formula:


58. The substrate of claim 41, wherein said substrate has the formula:


59. A substrate for detecting native enzyme activity in acidicorganelles comprising the formula:T-F(R)-BLOCK where; T represents a targeting group that is a weaklybasic amine containing compound that partitions the substrate to theacidic organelle; F represents a reporter that has further elaborationwith one or more substituents R to provide for a visible signal at lowpH values; BLOCK is a monovalent moiety adapted to be cleaved from theremainder of the substrate by action of a specific enzyme native to saidacidic organelle, resulting in F providing a visible signal at the siteof enzyme reaction.
 60. A method of measuring enzyme activity in acidicorgandies, comprising: a) combining a sample that comprises an isolatedacidic organelle with a biocompatible solution comprising a substrate ofthe formula:T-F(R)-BLOCK ; and b) detecting the presence of a visible signal;where 1) T represents a targeting group that is a weakly basic aminecontaining compound that partitions the substrate to the acidicorganelle; 2) F represents a reporter that has further elaboration withone or more substituents R to provide for a visible signal at low pHvalues; 3) BLOCK is a monovalent moiety adapted to be cleaved from theremainder of the substrate by action of a specific enzyme native to saidacidic organelle, resulting in F providing a visible signal at the siteof enzyme reaction; and 4) said sample and substrate are contacted for atime sufficient for said native enzyme to remove BLOCK and produce saidvisible signal.